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Sample records for cultured brain cells

  1. Novel culturing platform for brain slices and neuronal cells

    DEFF Research Database (Denmark)

    Svendsen, Winnie Edith; Al Atraktchi, Fatima Al-Zahraa; Bakmand, Tanya

    2015-01-01

    In this paper we demonstrate a novel culturing system for brain slices and neuronal cells, which can control the concentration of nutrients and the waste removal from the culture by adjusting the fluid flow within the device. The entire system can be placed in an incubator. The system has been te...... tested successfully with brain slices and PC12 cells. The culture substrate can be modified using metal electrodes and/or nanostructures for conducting electrical measurements while culturing and for better mimicking the in vivo conditions.......In this paper we demonstrate a novel culturing system for brain slices and neuronal cells, which can control the concentration of nutrients and the waste removal from the culture by adjusting the fluid flow within the device. The entire system can be placed in an incubator. The system has been...

  2. Generation of primary cultures of bovine brain endothelial cells and setup of cocultures with rat astrocytes

    DEFF Research Database (Denmark)

    Helms, Hans C; Brodin, Birger

    2014-01-01

    -brain barrier. The present protocol describes the setup of an in vitro coculture model based on primary cultures of endothelial cells from bovine brain microvessels and primary cultures of rat astrocytes. The model displays a high electrical tightness and expresses blood-brain barrier marker proteins....

  3. Transfection of brain capillary endothelial cells in primary culture with defined blood-brain barrier properties.

    Science.gov (United States)

    Burkhart, Annette; Thomsen, Louiza Bohn; Thomsen, Maj Schneider; Lichota, Jacek; Fazakas, Csilla; Krizbai, István; Moos, Torben

    2015-08-07

    Primary brain capillary endothelial cells (BCECs) are a promising tool to study the blood-brain barrier (BBB) in vitro, as they maintain many important characteristics of the BBB in vivo, especially when co-cultured with pericytes and/or astrocytes. A novel strategy for drug delivery to the brain is to transform BCECs into protein factories by genetic modifications leading to secretion of otherwise BBB impermeable proteins into the central nervous system. However, a huge challenge underlying this strategy is to enable transfection of non-mitotic BCECs, taking a non-viral approach. We therefore aimed to study transfection in primary, non-mitotic BCECs cultured with defined BBB properties without disrupting the cells' integrity. Primary cultures of BCECs, pericytes and astrocytes were generated from rat brains and used in three different in vitro BBB experimental arrangements, which were characterised based on a their expression of tight junction proteins and other BBB specific proteins, high trans-endothelial electrical resistance (TEER), and low passive permeability to radiolabeled mannitol. Recombinant gene expression and protein synthesis were examined in primary BCECs. The BCECs were transfected using a commercially available transfection agent Turbofect™ to express the red fluorescent protein HcRed1-C1. The BCECs were transfected at different time points to monitor transfection in relation to mitotic or non-mitotic cells, as indicated by fluorescence-activated cell sorting analysis after 5-and 6-carboxylfluorescein diacetate succinidyl ester incorporation. The cell cultures exhibited important BBB characteristics judged from their expression of BBB specific proteins, high TEER values, and low passive permeability. Among the three in vitro BBB models, co-culturing with BCECs and astrocytes was well suited for the transfection studies. Transfection was independent of cell division and with equal efficacy between the mitotic and non-mitotic BCECs. Importantly

  4. Culturing of PC12 Cells, Neuronal Cells, Astrocytes Cultures and Brain Slices in an Open Microfluidic System

    DEFF Research Database (Denmark)

    Al Atraktchi, Fatima Al-Zahraa; Bakmand, Tanya; Rømer Sørensen, Ane

    and electrochemical sensor system that enables real time detection of metabolites, e.g. dopamine from cell cultures and brain slices. In summary we present results on culturing of brain slices and cells in the microfluidic system as well as on the incorporation of an electrochemical sensor system for characterization......The brain is the center of the nervous system, where serious neurodegenerative diseases such as Parkinson’s, Alzheimer’s and Huntington’s are products of functional loss in the neural cells (1). Typical techniques used to investigate these diseases lack precise control of the cellular surroundings......, in addition to isolating the neural tissue from nutrient delivery and to creating unwanted gradients (2). This means that typical techniques used to investigate neurodegenerative diseases cannot mimic in vivo conditions, as closely as desired. We have developed a novel microfluidic system for culturing PC12...

  5. Quantitation of DNA repair in brain cell cultures: implications for autoradiographic analysis of mixed cell populations

    International Nuclear Information System (INIS)

    Dambergs, R.; Kidson, C.

    1979-01-01

    Quantitation of DNA repair in the mixed cell population of mouse embryo brain cultures has been assessed by autoradiographic analysis of unscheduled DNA synthesis following UV-irradiation. The proportion of labelled neurons and the grain density over neuronal nuclei were both less than the corresponding values for glial cells. The nuclear geometries of these two classes of cell are very different. Partial correction for the different geometries by relating grain density to nuclear area brought estimates of neuronal and glial DNA repair synthesis more closely in line. These findings have general implications for autoradiographic measurement of DNA repair in mixed cell populations and in differentiated versus dividing cells. (author)

  6. A porcine astrocyte/endothelial cell co-culture model of the blood-brain barrier.

    Science.gov (United States)

    Jeliazkova-Mecheva, Valentina V; Bobilya, Dennis J

    2003-10-01

    A method for the isolation of porcine atrocytes as a simple extension of a previously described procedure for isolation of brain capillary endothelial cells from adolescent pigs [Methods Cell Sci. 17 (1995) 2] is described. The obtained astroglial culture purified through two passages and by the method of the selective detachment was validated by a phase contrast microscopy and through an immunofluorescent assay for the glial fibrillary acidic protein (GFAP). Porcine astrocytes were co-cultivated with porcine brain capillary endothelial cells (PBCEC) for the development of an in vitro blood-brain barrier (BBB) model. The model was visualized by an electron microscopy and showed elevated transendothellial electrical resistance and reduced inulin permeability. To our knowledge, this is the first report for the establishment of a porcine astrocyte/endothelial cell co-culture BBB model, which avoids interspecies and age differences between the two cell types, usually encountered in the other reported co-culture BBB models. Considering the availability of the porcine brain tissue and the close physiological and anatomical relation between the human and pig brain, the porcine astrocyte/endothelial cell co-culture system can serve as a reliable and easily reproducible model for different in vitro BBB studies.

  7. Dopaminergic differentiation of human neural stem cells mediated by co-cultured rat striatal brain slices

    DEFF Research Database (Denmark)

    Anwar, Mohammad Raffaqat; Andreasen, Christian Maaløv; Lippert, Solvej Kølvraa

    2008-01-01

    differentiation, we co-cultured cells from a human neural forebrain-derived stem cell line (hNS1) with rat striatal brain slices. In brief, coronal slices of neonatal rat striatum were cultured on semiporous membrane inserts placed in six-well trays overlying monolayers of hNS1 cells. After 12 days of co......Properly committed neural stem cells constitute a promising source of cells for transplantation in Parkinson's disease, but a protocol for controlled dopaminergic differentiation is not yet available. To establish a setting for identification of secreted neural compounds promoting dopaminergic...

  8. An improved in vitro blood-brain barrier model: rat brain endothelial cells co-cultured with astrocytes.

    Science.gov (United States)

    Abbott, N Joan; Dolman, Diana E M; Drndarski, Svetlana; Fredriksson, Sarah M

    2012-01-01

    In vitro blood-brain barrier (BBB) models using primary cultured brain endothelial cells are important for establishing cellular and molecular mechanisms of BBB function. Co-culturing with BBB-associated cells especially astrocytes to mimic more closely the in vivo condition leads to upregulation of the BBB phenotype in the brain endothelial cells. Rat brain endothelial cells (RBECs) are a valuable tool allowing ready comparison with in vivo studies in rodents; however, it has been difficult to obtain pure brain endothelial cells, and few models achieve a transendothelial electrical resistance (TEER, measure of tight junction efficacy) of >200 Ω cm(2), i.e. the models are still relatively leaky. Here, we describe methods for preparing high purity RBECs and neonatal rat astrocytes, and a co-culture method that generates a robust, stable BBB model that can achieve TEER >600 Ω cm(2). The method is based on >20 years experience with RBEC culture, together with recent improvements to kill contaminating cells and encourage BBB differentiation.Astrocytes are isolated by mechanical dissection and cell straining and are frozen for later co-culture. RBECs are isolated from 3-month-old rat cortices. The brains are cleaned of meninges and white matter and enzymatically and mechanically dissociated. Thereafter, the tissue homogenate is centrifuged in bovine serum albumin to separate vessel fragments from other cells that stick to the myelin plug. The vessel fragments undergo a second enzyme digestion to separate pericytes from vessels and break down vessels into shorter segments, after which a Percoll gradient is used to separate capillaries from venules, arterioles, and single cells. To kill remaining contaminating cells such as pericytes, the capillary fragments are plated in puromycin-containing medium and RBECs grown to 50-60% confluence. They are then passaged onto filters for co-culture with astrocytes grown in the bottom of the wells. The whole procedure takes ∼2

  9. A Triple Culture Model of the Blood-Brain Barrier Using Porcine Brain Endothelial cells, Astrocytes and Pericytes.

    Science.gov (United States)

    Thomsen, Louiza Bohn; Burkhart, Annette; Moos, Torben

    2015-01-01

    In vitro blood-brain barrier (BBB) models based on primary brain endothelial cells (BECs) cultured as monoculture or in co-culture with primary astrocytes and pericytes are useful for studying many properties of the BBB. The BECs retain their expression of tight junction proteins and efflux transporters leading to high trans-endothelial electric resistance (TEER) and low passive paracellular permeability. The BECs, astrocytes and pericytes are often isolated from small rodents. Larger species as cows and pigs however, reveal a higher yield, are readily available and have a closer resemblance to humans, which make them favorable high-throughput sources for cellular isolation. The aim of the present study has been to determine if the preferable combination of purely porcine cells isolated from the 6 months old domestic pigs, i.e. porcine brain endothelial cells (PBECs) in co-culture with porcine astrocytes and pericytes, would compare with PBECs co-cultured with astrocytes and pericytes isolated from newborn rats with respect to TEER value and low passive permeability. The astrocytes and pericytes were grown both as contact and non-contact co-cultures as well as in triple culture to examine their effects on the PBECs for barrier formation as revealed by TEER, passive permeability, and expression patterns of tight junction proteins, efflux transporters and the transferrin receptor. This syngenic porcine in vitro BBB model is comparable to triple cultures using PBECs, rat astrocytes and rat pericytes with respect to TEER formation, low passive permeability, and expression of hallmark proteins signifying the brain endothelium (tight junction proteins claudin 5 and occludin, the efflux transporters P-glycoprotein (PgP) and breast cancer related protein (BCRP), and the transferrin receptor).

  10. Neural stem cells improve neuronal survival in cultured postmortem brain tissue from aged and Alzheimer patients

    NARCIS (Netherlands)

    Wu, L.; Sluiter, A.A.; Guo, Ho Fu; Balesar, R. A.; Swaab, D. F.; Zhou, Jiang Ning; Verwer, R. W H

    Neurodegenerative diseases are progressive and incurable and are becoming ever more prevalent. To study whether neural stem cell can reactivate or rescue functions of impaired neurons in the human aging and neurodegenerating brain, we co-cultured postmortem slices from Alzheimer patients and control

  11. Distinct angiotensin II receptor in primary cultures of glial cells from rat brain

    International Nuclear Information System (INIS)

    Raizada, M.K.; Phillips, M.I.; Crews, F.T.; Sumners, C.

    1987-01-01

    Angiotensin II (Ang-II) has profound effects on the brain. Receptors for Ang-II have been demonstrated on neurons, but no relationship between glial cells and Agn-II has been established. Glial cells (from the hypothalamus and brain stem of 1-day-old rat brains) in primary culture have been used to demonstrate the presence of specific Ang-II receptors. Binding of 125 I-Ang-II to glial cultures was rapid, reversible, saturable, and specific for Ang-II. The rank order of potency of 125 I-Ang-II binding was determined. Scatchard analysis revealed a homogeneous population of high-affinity binding sites with a B/sub max/ of 110 fmol/mg of protein. Light-microscopic autoradiography of 125 I-Ang-II binding supported the kinetic data, documenting specific Ang-II receptors on the glial cells. Ang-II stimulated a dose-dependent hydrolysis of phosphatidylinositols in glial cells, an effect mediated by Ang-II receptors. However, Ang-II failed to influence [ 3 H] norepinephrine uptake, and catecholamines failed to regulate Ang-II receptors, effects that occur in neurons. These observations demonstrate the presence of specific Ang-II receptors on the glial cells in primary cultures derived from normotensive rat brain. The receptors are kinetically similar to, but functionally distinct from, the neuronal Ang-II receptors

  12. Ultrastructural and biochemical findings in brain cell cultures infected with canine distemper virus.

    Science.gov (United States)

    Glaus, T; Griot, C; Richard, A; Althaus, U; Herschkowitz, N; Vandevelde, M

    1990-01-01

    To study the pathomechanism of demyelination in canine distemper (CD), dog brain cell cultures were infected with virulent A75/17-CD virus (CDV) and examined ultrastructurally. Special attention was paid to the oligodendrocytes, which were specifically immunolabelled. In addition, cerebroside sulfotransferase (CST), an enzyme specific for oligodendrocyte activity was assayed during the course of the infection. Infection and maturation as well as CDV-induced changes were found in astrocytes and brain macrophages. Infection of oligodendrocytes was rarely seen, although CST activity of the culture markedly decreased and vacuolar degeneration of these cells occurred, resulting in their complete disappearance. We concluded that the degeneration of oligodendrocytes and demyelination is not due to direct virus-oligodendrocyte interaction, but due to CDV-induced events in other glial cells.

  13. Transferrin, carbonic anhydrase C and ferritin in dissociated murine brain cell cultures.

    Science.gov (United States)

    Griot, C; Vandevelde, M

    1988-07-01

    It is shown here that transferrin (Tf), the iron transport protein and carbonic anhydrase C (CA C) are specifically located within oligodendrocytes in murine brain cell cultures. Ferritin (F), the major iron storage protein, was demonstrated in oligodendrocytes, as well as in astrocytes and microglial cells and was more prominent in the former. CA C and Tf were seen first after 6-7 days in culture. CA C and F positivity increased rapidly and at day 20, 80-85% of galactocerebroside + oligodendrocytes were positive for both proteins. Only a small number of oligodendrocytes was Tf+ up to day 14, after which their numbers increased rapidly until day 20, when 67% of the oligodendrocytes were Tf+. Because of the presence of Tf and F in oligodendrocytes it is suggested that these cells may play an important role in the metabolism of iron within the central nervous system.

  14. Lithium treatment elongates primary cilia in the mouse brain and in cultured cells

    Energy Technology Data Exchange (ETDEWEB)

    Miyoshi, Ko, E-mail: miyoshi@cc.okayama-u.ac.jp [Department of Brain Science, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 2-5-1 Shikatacho, Okayama 700-8558 (Japan); Kasahara, Kyosuke; Miyazaki, Ikuko; Asanuma, Masato [Department of Brain Science, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 2-5-1 Shikatacho, Okayama 700-8558 (Japan)

    2009-10-30

    The molecular mechanisms underlying the therapeutic effects of lithium, a first-line antimanic mood stabilizer, have not yet been fully elucidated. Treatment of the algae Chlamydomonas reinhardtii with lithium has been shown to induce elongation of their flagella, which are analogous structures to vertebrate cilia. In the mouse brain, adenylyl cyclase 3 (AC3) and certain neuropeptide receptors colocalize to the primary cilium of neuronal cells, suggesting a chemosensory function for the primary cilium in the nervous system. Here we show that lithium treatment elongates primary cilia in the mouse brain and in cultured cells. Brain sections from mice chronically fed with Li{sub 2}CO{sub 3} were subjected to immunofluorescence study. Primary cilia carrying both AC3 and the receptor for melanin-concentrating hormone (MCH) were elongated in the dorsal striatum and nucleus accumbens of lithium-fed mice, as compared to those of control animals. Moreover, lithium-treated NIH3T3 cells and cultured striatal neurons exhibited elongation of the primary cilia. The present results provide initial evidence that a psychotropic agent can affect ciliary length in the central nervous system, and furthermore suggest that lithium exerts its therapeutic effects via the upregulation of cilia-mediated MCH sensing. These findings thus contribute novel insights into the pathophysiology of bipolar mood disorder and other psychiatric diseases.

  15. Lithium treatment elongates primary cilia in the mouse brain and in cultured cells

    International Nuclear Information System (INIS)

    Miyoshi, Ko; Kasahara, Kyosuke; Miyazaki, Ikuko; Asanuma, Masato

    2009-01-01

    The molecular mechanisms underlying the therapeutic effects of lithium, a first-line antimanic mood stabilizer, have not yet been fully elucidated. Treatment of the algae Chlamydomonas reinhardtii with lithium has been shown to induce elongation of their flagella, which are analogous structures to vertebrate cilia. In the mouse brain, adenylyl cyclase 3 (AC3) and certain neuropeptide receptors colocalize to the primary cilium of neuronal cells, suggesting a chemosensory function for the primary cilium in the nervous system. Here we show that lithium treatment elongates primary cilia in the mouse brain and in cultured cells. Brain sections from mice chronically fed with Li 2 CO 3 were subjected to immunofluorescence study. Primary cilia carrying both AC3 and the receptor for melanin-concentrating hormone (MCH) were elongated in the dorsal striatum and nucleus accumbens of lithium-fed mice, as compared to those of control animals. Moreover, lithium-treated NIH3T3 cells and cultured striatal neurons exhibited elongation of the primary cilia. The present results provide initial evidence that a psychotropic agent can affect ciliary length in the central nervous system, and furthermore suggest that lithium exerts its therapeutic effects via the upregulation of cilia-mediated MCH sensing. These findings thus contribute novel insights into the pathophysiology of bipolar mood disorder and other psychiatric diseases.

  16. Responsiveness of fetal rat brain cells to glia maturation factor during neoplastic transformation in cell culture

    DEFF Research Database (Denmark)

    Haugen, A; Laerum, O D; Bock, E

    1981-01-01

    The effect of partially purified extracts from adult pig brains containing a glia maturation protein factor (BE) has been investigated on neural cells during carcinogenesis. Pregnant BD IX-rats were given a single transplacental dose of the carcinogen ethylnitrosourea (EtNU) on the 18th day...

  17. The toxicity of uranyl nitrate on primary brain cell culture of L. Hoevenii

    International Nuclear Information System (INIS)

    Ismail Bahari; Fauziah Mohd Noor

    1995-01-01

    In Malaysia, uranium is indirectly being concentrated by mining and petroleum industries that have no relevance to its use. Concentration of uranium and the production of TENORM may give rise to radiological risk to workers and the environment. A study was conducted to determine the toxicity of a uranium compound, uranyl nitrate. For this purpose a primary brain cell culture derived from L. hoevenii was used. The nature of uranil nitrate toxicity was determined by comparing with the effects induced by mitomycin C and gamma radiation. The toxicity of these agents were measured by observing changes in Unschedule DNA Synthesis (UDS) and the induction of micronucleus. Result from the study showed that UO sub 2 sup 2+ is UDS positive and is toxic to the primary brain cells of L. hoevenii. It gives a response profile that is almost similar to that induced by gamma radiation and mitomycin C. We believed that a low concentration, UO sub 2 sup 2+ acts as a chemo toxic agent rather than as an ionising radiation. At higher concentration the toxicity of UO sub 2 sup 2+ comes from both its chemo toxic and radiation effects. Results of this study also show the ability of the primary culture to carry out repair on its DNA damaged by the UDS positive agents

  18. Cell type-specific expression and localization of cytochrome P450 isoforms in tridimensional aggregating rat brain cell cultures.

    Science.gov (United States)

    Vichi, S; Sandström von Tobel, J; Gemma, S; Stanzel, S; Kopp-Schneider, A; Monnet-Tschudi, F; Testai, E; Zurich, M G

    2015-12-25

    Within the Predict-IV FP7 project a strategy for measurement of in vitro biokinetics was developed, requiring the characterization of the cellular model used, especially regarding biotransformation, which frequently depends on cytochrome P450 (CYP) activity. The extrahepatic in situ CYP-mediated metabolism is especially relevant in target organ toxicity. In this study, the constitutive mRNA levels and protein localization of different CYP isoforms were investigated in 3D aggregating brain cell cultures. CYP1A1, CYP2B1/B2, CYP2D2/4, CYP2E1 and CYP3A were expressed; CYP1A1 and 2B1 represented almost 80% of the total mRNA content. Double-immunolabeling revealed their presence in astrocytes, in neurons, and to a minor extent in oligodendrocytes, confirming the cell-specific localization of CYPs in the brain. These results together with the recently reported formation of an amiodarone metabolite following repeated exposure suggest that this cell culture system possesses some metabolic potential, most likely contributing to its high performance in neurotoxicological studies and support the use of this model in studying brain neurotoxicity involving mechanisms of toxication/detoxication. Copyright © 2015 Elsevier Ltd. All rights reserved.

  19. Apoptosis supression of cultured mouse brain cells by low dose rate irradiation of heavy particles

    International Nuclear Information System (INIS)

    Nagaoka, S.; Nojima, K.; Uchihori, Y.; Suzuki, M.

    2003-01-01

    An environment of low dose rate radiation of heavy particles was achieved in an incubator by using off-centered scattered beam of accelerator (HIMAC). Cultured hole brain cells prepared from newborn mice of C3H, SCID and B6 were incubated in 10% FBS-DMEM medium for five days in the HIMAC incubator irradiating the low dose rate of heavy particles. The heavy particle irradiation was passively occurred while the HIMAC beam port was activated during the other experiments in the night period. The radiation doses inside and outside the incubator were continuously measured by LIULIN detectors. The accumulated dose during incubation varied 0.5-10mGy depend on the frequency of HIMAC operations. The apoptotic sensitivities of the brain cells were evaluated by fixing and Hoechst33342 staining after additional acute exposure of 200 KV X-ray within 4 Gy. A slight increase in apoptotic fraction without the acute irradiation was observed in cells inside the incubator compared to the laboratory control. The apoptosis induction was however suppressed when the additional X-ray irradiation was applied in a range of 0.25- 0.75Gy. This suggested that an adaptive response may be induced when heavy particle irradiation of the low dose rate was applied prior to the acute X-ray irradiation. This paper will discuss effects of the accumulated doses of the preceding irradiation of heavy particles and periods until the subsequent acute irradiation

  20. Impact of commercial cigarette smoke condensate on brain tissue co-cultured with astrocytes and blood-brain barrier endothelial cells.

    Science.gov (United States)

    Lee, Seon-Bong; Kim, Ju-Hyeong; Cho, Myung-Haing; Choe, Eun-Sang; Kim, Kwang-Sik; Shim, Soon-Mi

    2017-01-01

    The purpose of the current study was to investigate the effect of two commercial cigarette smoke condensates (CCSC) on oxidative stress and cell cytotoxicity in human brain (T98G) or astrocytes (U-373 MG) in the presence of human brain microvascular endothelial cells (HBMEC). Cell viability of mono-culture of T98G or U-373 MG was markedly decreased in a concentration-dependent manner, and T98G was more susceptible than U-373 MG to CCSC exposure. Cytotoxicity was less prominent when T98G was co-cultured with HBMEC than when T98G was co-cultured with U-373 MG. Significant reduction in trans-epithelial electric resistance (TEER), a biomarker of cellular integrity was noted in HBMEC co-cultured with T98G (HBMEC-T98G co-culture) and U-373 MG co-cultured with T98G (U-373 MG-T98G co-culture) after 24 or 48 hr CCSC exposure, respectively. TEER value of U-373 MG co-cultured with T98G (79-84%) was higher than HBMEC co-cultured with T98G (62-63%) within 120-hr incubation with CCSC. Reactive oxygen species (ROS) generated by CCSC in mono-culture of T98G and U-373 MG reached highest levels at 4 and 16 mg/ml, respectively. ROS production by T98G fell when co-cultured with HBMEC or U-373MG. These findings suggest that adverse consequences of CCSC treatment on brain cells may be protected by blood-brain barrier or astrocytes, but with chronic exposure toxicity may be worsened due to destruction of cellular integrity.

  1. Regulation of cyclic AMP by extracellular ATP in cultured brain capillary endothelial cells

    Science.gov (United States)

    Anwar, Zubeya; Albert, Jennifer L; Gubby, Sharon E; Boyle, John P; Roberts, Jonathon A; Webb, Tania E; Boarder, Michael R

    1999-01-01

    In primary unpassaged rat brain capillary endothelial cell cultures (RBECs), using reverse-transcriptase PCR with primers specific for P2Y receptor subtypes, we detected mRNA for P2Y2, P2Y4 and P2Y6, but not P2Y1 receptors.None of the various nucleotides tested reduced forskolin elevated cyclic AMP levels in RBECs. ATP and ATPγS, as well as adenosine, enhanced cyclic AMP accumulation in the presence of forskolin.Comparison of the concentration response curves to ATPγS with those for ATP and adenosine, at different incubation times, indicated that the response to purine nucleotides was not wholly dependent on conversion to adenosine. Adenosine deaminase abolished the response to adenosine but only reduced the response to ATP by about 50%. These results suggest the participation of a receptor responsive to nucleotides.Isobutylmethylxanthine and 8-sulphophenyltheophylline prevented the cyclic AMP response, while neither 8-cyclopentyl-1,3-dipropylxanthine nor SCH58261 were effective antagonists. 2-chloradenosine gave a robust response, but neither 2-chloro-N6-cyclopentyladenosine nor CGS 21680 were agonists.These results show that adenosine and ATP can elevate the cyclic AMP levels of brain endothelial cells by acting on receptors which have a pharmacology apparently distinct from known P2Y and adenosine receptors. PMID:10510459

  2. Brain, body and culture

    DEFF Research Database (Denmark)

    Geertz, Armin W.

    2010-01-01

    This essay sketches out a biocultural theory of religion which is based on an expanded view of cognition that is anchored in brain and body (embrained and embodied), deeply dependent on culture (enculturated) and extended and distributed beyond the borders of individual brains. Such an approach...... uniquely accommodates contemporary cultural and neurobiological sciences. Since the challenge that the study of religion faces, in my opinion, is at the interstices of these sciences, I have tried to develop a theory of religion which acknowledges the fact. My hope is that the theory can be of use...

  3. Direct Signaling from Astrocytes to Neurons in Cultures of Mammalian Brain Cells

    Science.gov (United States)

    Nedergaard, Maiken

    1994-03-01

    Although astrocytes have been considered to be supportive, rather than transmissive, in the adult nervous system, recent studies have challenged this assumption by demonstrating that astrocytes possess functional neurotransmitter receptors. Astrocytes are now shown to directly modulate the free cytosolic calcium, and hence transmission characteristics, of neighboring neurons. When a focal electric field potential was applied to single astrocytes in mixed cultures of rat forebrain astrocytes and neurons, a prompt elevation of calcium occurred in the target cell. This in turn triggered a wave of calcium increase, which propagated from astrocyte to astrocyte. Neurons resting on these astrocytes responded with large increases in their concentration of cytosolic calcium. The gap junction blocker octanol attenuated the neuronal response, which suggests that the astrocytic-neuronal signaling is mediated through intercellular connections rather than synaptically. This neuronal response to local astrocytic stimulation may mediate local intercellular communication within the brain.

  4. Delta-9-tetrahydrocannabinol accumulation, metabolism and cell-type-specific adverse effects in aggregating brain cell cultures

    International Nuclear Information System (INIS)

    Monnet-Tschudi, Florianne; Hazekamp, Arno; Perret, Nicolas; Zurich, Marie-Gabrielle; Mangin, Patrice; Giroud, Christian; Honegger, Paul

    2008-01-01

    Despite the widespread use of Cannabis as recreational drug or as medicine, little is known about its toxicity. The accumulation, metabolism and toxicity of THC were analyzed 10 days after a single treatment, and after repeated exposures during 10 days. Mixed-cell aggregate cultures of fetal rat telencephalon were used as in vitro model, as well as aggregates enriched either in neurons or in glial cells. It was found that THC accumulated preferentially in neurons, and that glia-neuron interactions decreased THC accumulation. The quantification of 11-OH-THC and of THC-COOH showed that brain aggregates were capable of THC metabolism. No cell-type difference was found for the metabolite 11-OH-THC, whereas the THC-COOH content was higher in mixed-cell cultures. No cell death was found at THC concentrations of 2 μM in single treatment and of 1 μM and 2 μM in repeated treatments. Neurons, and particularly GABAergic neurons, were most sensitive to THC. Only the GABAergic marker was affected after the single treatment, whereas the GABAergic, cholinergic and astrocytic markers were decreased after the repeated treatments. JWH 015, a CB2 receptor agonist, showed effects similar to THC, whereas ACEA, a CB1 receptor agonist, had no effect. The expression of the cytokine IL-6 was upregulated 48 h after the single treatment with 5 μM of THC or JWH 015, whereas the expression of TNF-α remained unchanged. These results suggest that the adverse effects of THC were related either to THC accumulation or to cannabinoid receptor activation and associated with IL-6 upregulation

  5. [The persistence of the causative agent of amyotrophic leukospongiosis and the astrocyte reaction in monolayer cultures of brain cells].

    Science.gov (United States)

    Poleshchuk, N N; Kvacheva, Z B; Il'kevich, Iu G; Kolomiets, N D

    1990-01-01

    The amyotrophic leukospongiosis (AL) agent which is considered to be an unconventional virus was shown to replicate and amplify in non-neuronal monolayer brain cell cultures. The AL agent persistence was accompanied by complicated morphofunctional changes in astrocytes, some of them developing a specific cytodystrophic process. Phagocytosis in the infected astrocytes came to its end. The dose-dependent effect and selective sensitivity of these cells to the cytodestructive activity of AL was demonstrated. Astrocytes are regarded to be target cells serving as a reservoir for agent amplification.

  6. Dissociated cultures of newborn mouse brain

    International Nuclear Information System (INIS)

    Wiesmann, U.N.; Hofmann, K.; Burkhart, T.; Herschkowitz, N.

    1975-01-01

    The metabolism of 35 SO 4 -sulfated lipids and mucopolysaccharides was studied in dissociated brain cell cultures from newborn albino mouse brains. The cultures were maintained under an atmosphere of 40% O 2 and 5% CO 2 in apparent good health up to 30 days. Early morphological examination of the dissociated cells demonstrated an initial partial reaggregation of the cells, which later settled and became confluent bilayered cultures. Cell proliferation measured by DNA and protein determination, morphological differentiation and biochemical differentiation took place in the dissociated brain cell cultures analogous in some respects to the in vivo situation. A timed increase in the synthesis of a myelin precursor, cerebroside 35 SO 4 , was observed after 6 to 8 days in culture (DIC). A peak of cerebroside sulfate was evident at 17 DIC. No stable sulfatide was observed at any time. Protein-bound macromolecular 35 SO 4 -MPS was synthetized and secreted from the cells into the culture medium. Maximal synthesis and secretion occurred at 8 DIC. This culture system proves to be a useful model for studying some aspects of differentiation of brain cells under external conditions. (author)

  7. Regional Susceptibility to Domoic Acid in Primary Astrocyte Cells Cultured from the Brain Stem and Hippocampus

    Directory of Open Access Journals (Sweden)

    Olga M. Pulido

    2008-02-01

    Full Text Available Domoic acid is a marine biotoxin associated with harmful algal blooms and is the causative agent of amnesic shellfish poisoning in marine animals and humans. It is also an excitatory amino acid analog to glutamate and kainic acid which acts through glutamate receptors eliciting a very rapid and potent neurotoxic response. The hippocampus, among other brain regions, has been identified as a specific target site having high sensitivity to DOM toxicity. Histopathology evidence indicates that in addition to neurons, the astrocytes were also injured. Electron microscopy data reported in this study further supports the light microscopy findings. Furthermore, the effect of DOM was confirmed by culturing primary astrocytes from the hippocampus and the brain stem and subsequently exposing them to domoic acid. The RNA was extracted and used for biomarker analysis. The biomarker analysis was done for the early response genes including c-fos, c-jun, c-myc, Hsp-72; specific marker for the astrocytes- GFAP and the glutamate receptors including GluR 2, NMDAR 1, NMDAR 2A and B. Although, the astrocyte-GFAP and c-fos were not affected, c-jun and GluR 2 were down-regulated. The microarray analysis revealed that the chemokines / cytokines, tyrosine kinases (Trk, and apoptotic genes were altered. The chemokines that were up-regulated included - IL1-a, IL-1B, IL-6, the small inducible cytokine, interferon protein IP-10, CXC chemokine LIX, and IGF binding proteins. The Bax, Bcl-2, Trk A and Trk B were all downregulated. Interestingly, only the hippocampal astrocytes were affected. Our findings suggest that astrocytes may present a possible target for pharmacological interventions for the prevention and treatment of amnesic shellfish poisoning and for other brain pathologies involving excitotoxicity

  8. Choroid plexus epithelial monolayers – a cell culture model from porcine brain

    Directory of Open Access Journals (Sweden)

    Reichel Valeska

    2006-12-01

    Full Text Available Abstract Background The goal of the present study was to develop an in vitro choroid plexus (CP epithelial cell culture model for studying transport of protein-mediated drug secretion from blood to cerebrospinal fluid (CSF and vice versa. Methods Cells were isolated by mechanical and enzymatic treatment of freshly isolated porcine plexus tissue. Epithelial cell monolayers were grown and CSF secretion and transepithelial resistance were determined. The expression of f-actin as well as the choroid plexus marker protein transthyretin (TTR, were assessed. The expression of the export proteins p-glycoprotein (Pgp, Abcb1 and multidrug resistance protein 1 (Mrp1, Abcc1 was studied by RT-PCR, Western-blot and immunofluorescence techniques and their functional activity was assessed by transport and uptake experiments. Results Choroid plexus epithelial cells were isolated in high purity and grown to form confluent monolayers. Filter-grown monolayers displayed transendothelial resistance (TEER values in the range of 100 to 150 Ωcm2. Morphologically, the cells showed the typical net work of f-actin and expressed TTR at a high rate. The cultured cells were able to secrete CSF at a rate of 48.2 ± 4.6 μl/cm2/h over 2–3 hours. The ABC-export protein Mrp1 was expressed in the basolateral (blood-facing membranes of cell monolayers and intact tissue. P-glycoprotein showed only low expression within the apical (CSF directed membrane but was located more in sub-apical cell compartments. This finding was paralleled by the lack of directed excretion of p-glycoprotein substrates, verapamil and rhodamine 123. Conclusion It was demonstrated that CP epithelium can be isolated and cultured, with cells growing into intact monolayers, fully differentiating and with properties resembling the tissue in vivo. Thus, the established primary porcine CP model, allowing investigation of complex transport processes, can be used as a reliable tool for analysis of xenobiotic

  9. Antiviral antibodies stimulate production of reactive oxygen species in cultured canine brain cells infected with canine distemper virus.

    Science.gov (United States)

    Bürge, T; Griot, C; Vandevelde, M; Peterhans, E

    1989-06-01

    Canine distemper is characterized mainly by respiratory, enteric, and nervous symptoms. Infection of the central nervous system results in demyelination, to which inflammation has been shown to contribute significantly. It has been proposed that macrophages play a major role as effector cells in this process. We report that cultured dog brain cells contain a population of macrophages capable of producing reactive oxygen species as measured by luminol-dependent chemiluminescence. In cultures infected with canine distemper virus, a burst of reactive oxygen is triggered by antiviral antibody. This response depends on the presence of viral antigens on the surfaces of infected cells and is mediated by the interaction of antigen-bound antibody with Fc receptors on the macrophages. Since there is no evidence in vitro or in vivo that oligodendrocytes, the cells forming myelin, are infected, our observation supports the hypothesis that "innocent bystander killing" is important in demyelination caused by canine distemper virus. Reactive oxygen species released from macrophages may contribute to destruction of myelin.

  10. Alkaline phosphatase expression in cultured endothelial cells of aorta and brain microvessels: induction by interleukin-6-type cytokines and suppression by transforming growth factor betas.

    Science.gov (United States)

    Nakazato, H; Deguchi, M; Fujimoto, M; Fukushima, H

    1997-01-01

    Alkaline phosphatase (ALP) activity is markedly high in endothelial cells of the blood-brain barrier (BBB) type but absent from or low in those of the non-BBB type. Interleukin 6 (IL-6) has been identified as a glial cell line-derived factor that induces high ALP activity in cultured aortic endothelial cells. In the present study, we examined the effect of IL-6-type cytokines and transforming growth factor betas (TGF-betas) on ALP expression in cultures of calf pulmonary aortic endothelial (CPAE) cells and porcine brain microvascular endothelial (PBME) cells. Leukemia inhibitory factor, ciliary neurotrophic factor, and oncostatin M, which are known as IL-6-type cytokines, induced high ALP expression in the CPAE cells but not in the PBME cells. ALP levels in these cells were markedly suppressed by culture with TGF-betas. However, in cultured PBME cells, IL-6 and a derivative of cyclic adenosine monophosphate significantly increased ALP activity. Our findings raise the posibility that local concentrations of IL-6, IL-6-type cytokines, and TGF-betas affect the ALP levels in the endothelial cells of aorta and brain microvessels under normal development and also under inflammatory conditions.

  11. Cultured bovine brain capillary endothelial cells (BBCEC) - a blood-brain barrier model for studying the binding and internalization of insulin and insulin-like growth factor 1

    International Nuclear Information System (INIS)

    Keller, B.T.; Borchardt, R.T.

    1987-01-01

    Cultured bovine brain capillary endothelial cells (BBCEC) have previously been reported by their laboratory as a working model for studying nutrient and drug transport and metabolism at the blood-brain barrier. In the present study, they have utilized this culture system to investigate the binding and internalization of [ 125 I]-labelled insulin (INS) and insulin-like growth factor 1(IGF-1) by BBCEC. After 2 hrs at 23 0 C, the specific binding of INS and IGF-1 was 1.6% and 13.6%, respectively. At 37 0 C, the maximum specific binding was 0.9% for INS and 5.8% for IGF-1. Using an acid-wash technique to assess peptide internalization, it was observed that, at 37 0 C, approximately 60% of the bound INS rapidly became resistant to acid treatment, a value which was constant over 2 hr. With IGF-1, a similar proportion of the bound material, 62%, became resistant by 30 min, but subsequently decreased to 45% by 2 hr. Scatchard analysis of competitive binding studies indicated the presence of two binding sites for each protein, having K/sub d/'s of 0.82 nM and 19.2 nM for INS and 0.39 nM and 3.66 nM for IGF-1. Little change in the amount of INS binding was observed over a four-day interval as the cultures became a confluent monolayer. The present report of binding and internalization of these proteins suggests that the BBCEC may utilize a receptor-mediated process to internalize and/or transport (transcytosis) INS and IGF-1 from the circulation

  12. A brain slice culture model for studies of endogenous and exogenous precursor cell migration in the rostral migratory stream

    DEFF Research Database (Denmark)

    Tanvig, Mette; Blaabjerg, Morten; Andersen, Rikke K

    2009-01-01

    week old cultures. For testing the migratory abilities of exogenous precursor cells, rat SVZ neurospheres and human neural (HNS1 cells) and mesenchymal (hMSC-TERT) stem cell lines were micrografted to the rostral SVZ of 1 and 7 day old cultures. Two weeks later graft derivatives were identified...

  13. Cultured cells of the blood-brain barrier from apolipoprotein B-100 transgenic mice: effects of oxidized low-density lipoprotein treatment.

    Science.gov (United States)

    Lénárt, Nikolett; Walter, Fruzsina R; Bocsik, Alexandra; Sántha, Petra; Tóth, Melinda E; Harazin, András; Tóth, Andrea E; Vizler, Csaba; Török, Zsolt; Pilbat, Ana-Maria; Vígh, László; Puskás, László G; Sántha, Miklós; Deli, Mária A

    2015-07-17

    The apolipoprotein B-100 (ApoB-100) transgenic mouse line is a model of human atherosclerosis. Latest findings suggest the importance of ApoB-100 in the development of neurodegenerative diseases and microvascular/perivascular localization of ApoB-100 protein was demonstrated in the cerebral cortex of ApoB-100 transgenic mice. The aim of the study was to characterize cultured brain endothelial cells, pericytes and glial cells from wild-type and ApoB-100 transgenic mice and to study the effect of oxidized low-density lipoprotein (oxLDL) on these cells. Morphology of cells isolated from brains of wild type and ApoB-100 transgenic mice was characterized by immunohistochemistry and the intensity of immunolabeling was quantified by image analysis. Toxicity of oxLDL treatment was monitored by real-time impedance measurement and lactate dehydrogenase release. Reactive oxygen species and nitric oxide production, barrier permeability in triple co-culture blood-brain barrier model and membrane fluidity were also determined after low-density lipoprotein (LDL) or oxLDL treatment. The presence of ApoB-100 was confirmed in brain endothelial cells, while no morphological change was observed between wild type and transgenic cells. Oxidized but not native LDL exerted dose-dependent toxicity in all three cell types, induced barrier dysfunction and increased reactive oxygen species (ROS) production in both genotypes. A partial protection from oxLDL toxicity was seen in brain endothelial and glial cells from ApoB-100 transgenic mice. Increased membrane rigidity was measured in brain endothelial cells from ApoB-100 transgenic mice and in LDL or oxLDL treated wild type cells. The morphological and functional properties of cultured brain endothelial cells, pericytes and glial cells from ApoB-100 transgenic mice were characterized and compared to wild type cells for the first time. The membrane fluidity changes in ApoB-100 transgenic cells related to brain microvasculature indicate

  14. Simultaneous quantification of monoamine neurotransmitters and their biogenic metabolites intracellularly and extracellularly in primary neuronal cell cultures and in sub-regions of guinea pig brain

    DEFF Research Database (Denmark)

    Schou-Pedersen, Anne Marie Voigt; Hansen, Stine Normann; Tveden-Nyborg, Pernille

    2016-01-01

    In the present paper, we describe a validated chromatographic method for the simultaneous quantification of monoamine neurotransmitters and their biogenic metabolites intracellularly and extracellularly in primary neuronal cell culture and in sub-regions of the guinea pig brain. Electrochemical...... of intracellular and extracellular amounts of monoamine neurotransmitters and their metabolites in guinea pig frontal cortex and hippocampal primary neuronal cell cultures. Noradrenaline, dopamine and serotonin were found to be in a range from 0.31 to 1.7 pmol per 2 million cells intracellularly, but only...

  15. Brain stem slice conditioned medium contains endogenous BDNF and GDNF that affect neural crest boundary cap cells in co-culture.

    Science.gov (United States)

    Kaiser, Andreas; Kale, Ajay; Novozhilova, Ekaterina; Siratirakun, Piyaporn; Aquino, Jorge B; Thonabulsombat, Charoensri; Ernfors, Patrik; Olivius, Petri

    2014-05-30

    Conditioned medium (CM), made by collecting medium after a few days in cell culture and then re-using it to further stimulate other cells, is a known experimental concept since the 1950s. Our group has explored this technique to stimulate the performance of cells in culture in general, and to evaluate stem- and progenitor cell aptitude for auditory nerve repair enhancement in particular. As compared to other mediums, all primary endpoints in our published experimental settings have weighed in favor of conditioned culture medium, where we have shown that conditioned culture medium has a stimulatory effect on cell survival. In order to explore the reasons for this improved survival we set out to analyze the conditioned culture medium. We utilized ELISA kits to investigate whether brain stem (BS) slice CM contains any significant amounts of brain-derived neurotrophic factor (BDNF) and glial cell derived neurotrophic factor (GDNF). We further looked for a donor cell with progenitor characteristics that would be receptive to BDNF and GDNF. We chose the well-documented boundary cap (BC) progenitor cells to be tested in our in vitro co-culture setting together with cochlear nucleus (CN) of the BS. The results show that BS CM contains BDNF and GDNF and that survival of BC cells, as well as BC cell differentiation into neurons, were enhanced when BS CM were used. Altogether, we conclude that BC cells transplanted into a BDNF and GDNF rich environment could be suitable for treatment of a traumatized or degenerated auditory nerve. Copyright © 2014 Elsevier B.V. All rights reserved.

  16. Detergent resistant membrane-associated IDE in brain tissue and cultured cells: Relevance to Aβ and insulin degradation

    Directory of Open Access Journals (Sweden)

    Castaño Eduardo M

    2008-12-01

    Full Text Available Abstract Background Insulin degrading enzyme (IDE is implicated in the regulation of amyloid β (Aβ steady-state levels in the brain, and its deficient expression and/or activity may be a risk factor in sporadic Alzheimer's disease (AD. Although IDE sub-cellular localization has been well studied, the compartments relevant to Aβ degradation remain to be determined. Results Our results of live immunofluorescence, immuno gold electron-microscopy and gradient fractionation concurred to the demonstration that endogenous IDE from brain tissues and cell cultures is, in addition to its other localizations, a detergent-resistant membrane (DRM-associated metallopeptidase. Our pulse chase experiments were in accordance with the existence of two pools of IDE: the cytosolic one with a longer half-life and the membrane-IDE with a faster turn-over. DRMs-associated IDE co-localized with Aβ and its distribution (DRMs vs. non-DRMs and activity was sensitive to manipulation of lipid composition in vitro and in vivo. When IDE was mis-located from DRMs by treating cells with methyl-β-cyclodextrin (MβCD, endogenous Aβ accumulated in the extracellular space and exogenous Aβ proteolysis was impaired. We detected a reduced amount of IDE in DRMs of membranes isolated from mice brain with endogenous reduced levels of cholesterol (Chol due to targeted deletion of one seladin-1 allele. We confirmed that a moderate shift of IDE from DRMs induced a substantial decrement on IDE-mediated insulin and Aβ degradation in vitro. Conclusion Our results support the notion that optimal substrate degradation by IDE may require its association with organized-DRMs. Alternatively, DRMs but not other plasma membrane regions, may act as platforms where Aβ accumulates, due to its hydrophobic properties, reaching local concentration close to its Km for IDE facilitating its clearance. Structural integrity of DRMs may also be required to tightly retain insulin receptor and IDE for

  17. Monocrotophos induces the expression and activity of xenobiotic metabolizing enzymes in pre-sensitized cultured human brain cells.

    Directory of Open Access Journals (Sweden)

    Vinay K Tripathi

    Full Text Available The expression and metabolic profile of cytochrome P450s (CYPs is largely missing in human brain due to non-availability of brain tissue. We attempted to address the issue by using human brain neuronal (SH-SY5Y and glial (U373-MG cells. The expression and activity of CYP1A1, 2B6 and 2E1 were carried out in the cells exposed to CYP inducers viz., 3-methylcholanthrene (3-MC, cyclophosphamide (CPA, ethanol and known neurotoxicant- monocrotophos (MCP, a widely used organophosphorous pesticide. Both the cells show significant induction in the expression and CYP-specific activity against classical inducers and MCP. The induction level of CYPs was comparatively lower in MCP exposed cells than cells exposed to classical inducers. Pre-exposure (12 h of cells to classical inducers significantly added the MCP induced CYPs expression and activity. The findings were concurrent with protein ligand docking studies, which show a significant modulatory capacity of MCP by strong interaction with CYP regulators-CAR, PXR and AHR. Similarly, the known CYP inducers- 3-MC, CPA and ethanol have also shown significantly high docking scores with all the three studied CYP regulators. The expression of CYPs in neuronal and glial cells has suggested their possible association with the endogenous physiology of the brain. The findings also suggest the xenobiotic metabolizing capabilities of these cells against MCP, if received a pre-sensitization to trigger the xenobiotic metabolizing machinery. MCP induced CYP-specific activity in neuronal cells could help in explaining its effect on neurotransmission, as these CYPs are known to involve in the synthesis/transport of the neurotransmitters. The induction of CYPs in glial cells is also of significance as these cells are thought to be involved in protecting the neurons from environmental insults and safeguard them from toxicity. The data provide better understanding of the metabolizing capability of the human brain cells against

  18. Insulin: its binding to specific receptors and its stimulation of DNA synthesis and 2',3'-cyclic nucleotide phosphohydrolase in embryonic mouse brain cell cultures

    International Nuclear Information System (INIS)

    Shanker, G.; Pieringer, R.A.

    1986-01-01

    Previously, the authors demonstrated that ornithine decarboxylase was stimulated by insulin in cultures of embryonic mouse brain cells. In the present work, they have investigated the presence and specificity of insulin receptors in these cultures. A time study showed that maximum binding of 125 [I] labelled insulin was around 75 min. Other studies measured the influence of concentration and age on insulin binding. A displacement study using increasing concentrations of cold insulin, glucagon or growth hormone demonstrated that the specificity of the receptors for insulin was rather high. It was also found that insulin displayed a clear dose-dependent stimulation of thymidine incorporation into the brain cells. Insulin also stimulated the glial enzyme 2':3'-cyclic nucleotide phosphohydrolase (CNP-ase). The results suggest a dual role for insulin; it regulates both cell proliferation as well as differentiation

  19. Hypoxia-cultured human adipose-derived mesenchymal stem cells are non-oncogenic and have enhanced viability, motility, and tropism to brain cancer.

    Science.gov (United States)

    Feng, Y; Zhu, M; Dangelmajer, S; Lee, Y M; Wijesekera, O; Castellanos, C X; Denduluri, A; Chaichana, K L; Li, Q; Zhang, H; Levchenko, A; Guerrero-Cazares, H; Quiñones-Hinojosa, A

    2014-12-11

    Adult human adipose-derived mesenchymal stem cells (hAMSCs) are multipotent cells, which are abundant, easily collected, and bypass the ethical concerns that plague embryonic stem cells. Their utility and accessibility have led to the rapid development of clinical investigations to explore their autologous and allogeneic cellular-based regenerative potential, tissue preservation capabilities, anti-inflammatory properties, and anticancer properties, among others. hAMSCs are typically cultured under ambient conditions with 21% oxygen. However, physiologically, hAMSCs exist in an environment of much lower oxygen tension. Furthermore, hAMSCs cultured in standard conditions have shown limited proliferative and migratory capabilities, as well as limited viability. This study investigated the effects hypoxic culture conditions have on primary intraoperatively derived hAMSCs. hAMSCs cultured under hypoxia (hAMSCs-H) remained multipotent, capable of differentiation into osteogenic, chondrogenic, and adipogenic lineages. In addition, hAMSCs-H grew faster and exhibited less cell death. Furthermore, hAMSCs-H had greater motility than normoxia-cultured hAMSCs and exhibited greater homing ability to glioblastoma (GBM) derived from brain tumor-initiating cells from our patients in vitro and in vivo. Importantly, hAMSCs-H did not transform into tumor-associated fibroblasts in vitro and were not tumorigenic in vivo. Rather, hAMSCs-H promoted the differentiation of brain cancer cells in vitro and in vivo. These findings suggest an alternative culturing technique that can enhance the function of hAMSCs, which may be necessary for their use in the treatment of various pathologies including stroke, myocardial infarction, amyotrophic lateral sclerosis, and GBM.

  20. Transport of monocarboxylic acids at the blood-brain barrier: Studies with monolayers of primary cultured bovine brain capillary endothelial cells

    International Nuclear Information System (INIS)

    Terasaki, T.; Takakuwa, S.; Moritani, S.; Tsuji, A.

    1991-01-01

    The kinetics and mechanism of the transport of monocarboxylic acids (MCAs) were studied by using primary cultured bovine brain capillary endothelial cells. Concentration-dependent uptake of acetic acid was observed, and the kinetic parameters were estimated as follows: the Michaelis constant, Kt, was 3.41 ± 1.87 mM, the maximum uptake rate, Jmax, was 144.7 ± 55.7 nmol/mg of protein/min and the nonsaturable first-order rate constant, Kd, was 6.66 ± 1.98 microliters/mg of protein/min. At medium pH below 7.0, the uptake rate of [3H]acetic acid increased markedly with decreasing medium pH, whereas pH-independent uptake was observed in the presence of 10 mM acetic acid. An energy requirement for [3H]acetic acid uptake was also demonstrated, because metabolic inhibitors (2,4-dinitrophenol and rotenone) reduced significantly the uptake rate (P less than .05). Carbonylcyanide-p-trifluoro-methoxyphenylhydrazone, a protonophore, inhibited significantly the uptake of [3H]acetic acid at medium pH of 5.0 and 6.0, whereas 4,4'-diisothiocyanostilben-2,2'-disulfonic acid did not. Several MCAs inhibited significantly the uptake rate of [3H]acetic acid, whereas di- and tricarboxylic acids did not. The uptake of [3H]acetic acid was competitively inhibited by salicylic acid, with an inhibition constant, Ki, of 3.60 mM, suggesting a common transport system between acetic acid and salicylic acid. Moreover, at the medium pH of 7.4, salicylic acid and valproic acid inhibited significantly the uptake of [3H]acetic acid, demonstrating that the transport of MCA drugs could also be ascribed to the MCA transport system at the physiologic pH

  1. Modeling the ischemic blood-brain barrier; the effects of oxygen-glucose deprivation (OGD) on endothelial cells in culture

    DEFF Research Database (Denmark)

    Tornabene, Erica; Helms, Hans Christian Cederberg; Berndt, Philipp

    their passage through the capillary endothelium. An ischemic insult and the subsequent restoration of blood flow, defined as reperfusion, dramatically impair the BBB integrity, resulting in increased BBB permeability, modified transport pathways, edema and tissue damage. A deeper understanding of the permeation......Introduction - The blood-brain barrier (BBB) is a physical, transport and metabolic barrier which plays a key role in preventing uncontrolled exchanges between blood and brain, ensuring an optimal environment for neurons activity. This extent interface is created by the endothelial cells forming...... the wall of brain capillaries. The restrictive nature of the BBB is due to the tight junctions (TJs), which seal the intercellular clefts, limiting the paracellular diffusion, efflux transporters, which extrude xenobiotics, and metabolizing enzymes, which may break down or convert molecules during...

  2. Simultaneous quantification of monoamine neurotransmitters and their biogenic metabolites intracellularly and extracellularly in primary neuronal cell cultures and in sub-regions of guinea pig brain.

    Science.gov (United States)

    Schou-Pedersen, Anne Marie V; Hansen, Stine N; Tveden-Nyborg, Pernille; Lykkesfeldt, Jens

    2016-08-15

    In the present paper, we describe a validated chromatographic method for the simultaneous quantification of monoamine neurotransmitters and their biogenic metabolites intracellularly and extracellularly in primary neuronal cell culture and in sub-regions of the guinea pig brain. Electrochemical detection provided limits of quantifications (LOQs) between 3.6 and 12nM. Within the linear range, obtained recoveries were from 90.9±9.9 to 120±14% and intra-day and inter-day precisions found to be less than 5.5% and 12%, respectively. The analytical method was applicable for quantification of intracellular and extracellular amounts of monoamine neurotransmitters and their metabolites in guinea pig frontal cortex and hippocampal primary neuronal cell cultures. Noradrenaline, dopamine and serotonin were found to be in a range from 0.31 to 1.7pmol per 2 million cells intracellularly, but only the biogenic metabolites could be detected extracellularly. Distinct differences in monoamine concentrations were observed when comparing concentrations in guinea pig frontal cortex and cerebellum tissue with higher amounts of dopamine and its metabolites, 3,4-dihydroxyphenylacetic acid and homovanillic acid in frontal cortex, as compared to cerebellum. The chemical turnover in frontal cortex tissue of guinea pig was for serotonin successfully predicted from the turnover observed in the frontal cortex cell culture. In conclusion, the present analytical method shows high precision, accuracy and sensitivity and is broadly applicable to monoamine measurements in cell cultures as well as brain biopsies from animal models used in preclinical neurochemistry. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. Adaptation of tick-borne encephalitis virus from human brain to different cell cultures induces multiple genomic substitutions.

    Science.gov (United States)

    Ponomareva, Eugenia P; Ternovoi, Vladimir A; Mikryukova, Tamara P; Protopopova, Elena V; Gladysheva, Anastasia V; Shvalov, Alexander N; Konovalova, Svetlana N; Chausov, Eugene V; Loktev, Valery B

    2017-10-01

    The C11-13 strain from the Siberian subtype of tick-borne encephalitis virus (TBEV) was isolated from human brain using pig embryo kidney (PEK), 293, and Neuro-2a cells. Analysis of the complete viral genome of the C11-13 variants during six passages in these cells revealed that the cell-adapted C11-13 variants had multiple amino acid substitutions as compared to TBEV from human brain. Seven out of eight amino acids substitutions in the high-replicating C11-13(PEK) variant mapped to non-structural proteins; 13 out of 14 substitutions in the well-replicating C11-13(293) variant, and all four substitutions in the low-replicating C11-13(Neuro-2a) variant were also localized in non-structural proteins, predominantly in the NS2a (2), NS3 (6) and NS5 (3) proteins. The substitutions NS2a 1067 (Asn → Asp), NS2a 1168 (Leu → Val) in the N-terminus of NS2a and NS3 1745 (His → Gln) in the helicase domain of NS3 were found in all selected variants. We postulate that multiple substitutions in the NS2a, NS3 and NS5 genes play a key role in adaptation of TBEV to different cells.

  4. Characterisation of an in vitro blood-brain barrier model based on primary porcine capillary endothelial cells in monoculture or co-culture with primary rat or porcine astrocytes and pericytes

    DEFF Research Database (Denmark)

    Thomsen, Louiza Bohn; Larsen, Annette Burkhart; Moos, Torben

    to in vivo such as efflux transporters, tight junction proteins, and high transendothelial electric resistance (TEER). Primary BCECs are isolated from a variety of mammals such as rats, mice, cattle and pigs. Often bovine and porcine BCECs are cultured in monoculture or in co-culture with rat astrocytes......In vitro blood-brain barrier (BBB) models based on primary brain capillary endothelial cells (BCECs) in monoculture or in co-culture with primary astrocytes and pericytes are often applied for studying physiology of the BBB. Primary BCECs retain many morphological and biochemical properties similar...... obtained from neonatal rats which have been shown to strengthen the barrier properties of the BCECs. In this study, brain endothelial cells (PBECs), astrocytes and pericytes are isolated from pig brains donated by the local abattoir. The brains are from 6 month old domestic pigs. The availability and high...

  5. Establishment of a Human Blood-Brain Barrier Co-culture Model Mimicking the Neurovascular Unit Using Induced Pluri- and Multipotent Stem Cells.

    Science.gov (United States)

    Appelt-Menzel, Antje; Cubukova, Alevtina; Günther, Katharina; Edenhofer, Frank; Piontek, Jörg; Krause, Gerd; Stüber, Tanja; Walles, Heike; Neuhaus, Winfried; Metzger, Marco

    2017-04-11

    In vitro models of the human blood-brain barrier (BBB) are highly desirable for drug development. This study aims to analyze a set of ten different BBB culture models based on primary cells, human induced pluripotent stem cells (hiPSCs), and multipotent fetal neural stem cells (fNSCs). We systematically investigated the impact of astrocytes, pericytes, and NSCs on hiPSC-derived BBB endothelial cell function and gene expression. The quadruple culture models, based on these four cell types, achieved BBB characteristics including transendothelial electrical resistance (TEER) up to 2,500 Ω cm 2 and distinct upregulation of typical BBB genes. A complex in vivo-like tight junction (TJ) network was detected by freeze-fracture and transmission electron microscopy. Treatment with claudin-specific TJ modulators caused TEER decrease, confirming the relevant role of claudin subtypes for paracellular tightness. Drug permeability tests with reference substances were performed and confirmed the suitability of the models for drug transport studies. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  6. Bacterial cell culture

    OpenAIRE

    sprotocols

    2014-01-01

    ### Materials 1. Glass culture tubes with metal caps and labels - Growth medium, from media room or customized - Glass pipette tubes - Parafilm ### Equipment 1. Vortexer - Fireboy or Bunsen burner - Motorized pipette - Micropipettes and sterile tips ### Procedure For a typical liquid culture, use 5 ml of appropriate medium. The amount in each tube does not have to be exact if you are just trying to culture cells for their precious DNA. 1. Streak an a...

  7. Basic cell culture.

    Science.gov (United States)

    Pollard, J W

    1990-01-01

    This article will describe the basic techniques required for successful cell culture. It will also act to introduce some of the other chapters in this volume. It is not intended, as this volume is not, to describe the establishment of a tissue culture laboratory, nor to provide a historical or theoretical survey of cell culture. There are several books that adequately cover these areas, including the now somewhat dated but still valuable volume by Paul (1), the multi-authored Methods in Enzymology volume edited by Jakoby and Pastan (2), and the new edition of Freshney (3). Instead, this chapter's focus will be on the techniques for establishing primary rodent cell cultures from embryos and adult skin, maintaining and subculturing these fibro-blasts and their transformed derivatives, and the isolation of genetically pure strains. The cells described are all derived from Chinese hamsters since, to date, these cells, have proved to be the most useful for somatic cell genetics (4,5). The techniques, however, are generally applicable to most fibroblastic cell types.

  8. Tissue cultures from adult human postmortem subcortical brain areas

    NARCIS (Netherlands)

    Verwer, R. W. H.; Dubelaar, E. J. G.; Hermens, W. T. J. M. C.; Swaab, D. F.

    2002-01-01

    Animal models used to study human aging and neurodegeneration do not display all symptoms of these processes as they are found in humans. Recently, we have shown that many cells in neocortical slices from adult human postmortem brain may survive for extensive periods in vitro. Such cultures may

  9. Cell Culturing of Cytoskeleton

    Science.gov (United States)

    2004-01-01

    Biomedical research offers hope for a variety of medical problems, from diabetes to the replacement of damaged bone and tissues. Bioreactors, which are used to grow cells and tissue cultures, play a major role in such research and production efforts. Cell culturing, such as this bone cell culture, is an important part of biomedical research. The BioDyn payload includes a tissue engineering investigation. The commercial affiliate, Millenium Biologix, Inc., has been conducting bone implant experiments to better understand how synthetic bone can be used to treat bone-related illnesses and bone damaged in accidents. On STS-95, the BioDyn payload will include a bone cell culture aimed to help develop this commercial synthetic bone product. Millenium Biologix, Inc., is exploring the potential for making human bone implantable materials by seeding its proprietary artificial scaffold material with human bone cells. The product of this tissue engineering experiment using the Bioprocessing Modules (BPMs) on STS-95 is space-grown bone implants, which could have potential for dental implants, long bone grafts, and coating for orthopedic implants such as hip replacements.

  10. Oscillating Cell Culture Bioreactor

    Science.gov (United States)

    Freed, Lisa E.; Cheng, Mingyu; Moretti, Matteo G.

    2010-01-01

    To better exploit the principles of gas transport and mass transport during the processes of cell seeding of 3D scaffolds and in vitro culture of 3D tissue engineered constructs, the oscillatory cell culture bioreactor provides a flow of cell suspensions and culture media directly through a porous 3D scaffold (during cell seeding) and a 3D construct (during subsequent cultivation) within a highly gas-permeable closed-loop tube. This design is simple, modular, and flexible, and its component parts are easy to assemble and operate, and are inexpensive. Chamber volume can be very low, but can be easily scaled up. This innovation is well suited to work with different biological specimens, particularly with cells having high oxygen requirements and/or shear sensitivity, and different scaffold structures and dimensions. The closed-loop changer is highly gas permeable to allow efficient gas exchange during the cell seeding/culturing process. A porous scaffold, which may be seeded with cells, is fixed by means of a scaffold holder to the chamber wall with scaffold/construct orientation with respect to the chamber determined by the geometry of the scaffold holder. A fluid, with/without biological specimens, is added to the chamber such that all, or most, of the air is displaced (i.e., with or without an enclosed air bubble). Motion is applied to the chamber within a controlled environment (e.g., oscillatory motion within a humidified 37 C incubator). Movement of the chamber induces relative motion of the scaffold/construct with respect to the fluid. In case the fluid is a cell suspension, cells will come into contact with the scaffold and eventually adhere to it. Alternatively, cells can be seeded on scaffolds by gel entrapment prior to bioreactor cultivation. Subsequently, the oscillatory cell culture bioreactor will provide efficient gas exchange (i.e., of oxygen and carbon dioxide, as required for viability of metabolically active cells) and controlled levels of fluid

  11. Culture of Mouse Neural Stem Cell Precursors

    OpenAIRE

    Currle, D. Spencer; Hu, Jia Sheng; Kolski-Andreaco, Aaron; Monuki, Edwin S.

    2007-01-01

    Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase our understanding of the nervous system and may allow us to develop treatments for currently incurable brain diseases and injuries. In addition, stem cells should be used for stem cell research aimed at the detailed study of mechanisms of neural differentiation and transdifferentiation and the genetic and environmental signals that direct the...

  12. Culture: by the brain and in the brain?

    Science.gov (United States)

    Ortega, Francisco; Vidal, Fernando

    2016-01-01

    Since the 1990s, several disciplines have emerged at the interface between neuroscience and the social and human sciences. For the most part, they aim at capturing the commonalities that underlay the heterogeneity of human behaviors and experiences. Neuroanthropology and cultural neuroscience, or the "neurodisciplines of culture," appear different, since their goal is to understand specificity rather than commonality and to address how cultural differences are inscribed in the brain. After offering an overview of these disciplines, and of their relation to endeavors such as cultural psychology and social neuroscience, this article discusses some of the most representative studies in the area in order to explore in which ways they are relevant for an understanding of culture.

  13. Long-term brain slice culturing in a microfluidic platform

    DEFF Research Database (Denmark)

    Vedarethinam, Indumathi; Avaliani, N.; Tønnesen, J.

    2011-01-01

    In this work, we present the development of a transparent poly(methyl methacrylate) (PMMA) based microfluidic culture system for handling long-term brain slice cultures independent of an incubator. The different stages of system development have been validated by culturing GFP producing brain...... brain slice culturing for 16 days....

  14. The blood-brain barrier in vitro using primary culture

    DEFF Research Database (Denmark)

    Larsen, Annette Burkhart

    of the thesis involves the establishment and characterization of an in vitro BBB models based on primary cells isolated from the rat brain. Co-culture and triple culture models with astrocytes and pericytes were found to be the superior to mono cultured BCECs with respect to many important BBB characteristics...... obstacle for the treatment of central nervous system (CNS) diseases, as many potentially CNS active drugs are unable to reach their site of action within the brain. In vitro BBB models are, therefore, being developed to investigate the BBB permeability of a drug early in its development. The first part....... In the second part of the thesis, the ability of turning BCECs into protein factories is investigated using a non-viral gene carrier. Transfection and protein synthesis of BCECs cultured with confined BBB properties were found to be feasible without disrupting the BBB properties, although it was not possible...

  15. Epithelial Cell Cultures

    Directory of Open Access Journals (Sweden)

    Imran S. Chaudhry

    2011-01-01

    Full Text Available The biological effects of only a finite number of tobacco toxins have been studied. Here, we describe exposure of cultures of human bronchial epithelial cells to low concentrations of tobacco carcinogens: nickel sulphate, benzo(bfluoranthene, N-nitrosodiethylamine, and 4-(methylnitrosamino-1-(3-pyridyl-1-butanone (NNK. After a 24-hour exposure, EGFR was expressed in cell membrane and cytoplasm, BCL-2 was expressed only in the irregular nuclei of large atypical cells, MKI67 was expressed in nuclei with no staining in larger cells, cytoplasmic BIRC5 with stronger nuclear staining was seen in large atypical cells, and nuclear TP53 was strongly expressed in all cells. After only a 24-hour exposure, cells exhibited atypical nuclear and cytoplasmic features. After a 48-hour exposure, EGFR staining was localized to the nucleus, BCL-2 was slightly decreased in intensity, BIRC5 was localized to the cytoplasm, and TP53 staining was increased in small and large cells. BCL2L1 was expressed in both the cytoplasm and nuclei of cells at 24- and 48-hour exposures. We illustrate that short-termexposure of a bronchial epithelial cell line to smoking-equivalent concentrations of tobacco carcinogens alters the expression of key proliferation regulatory genes, EGFR, BCL-2, BCL2L1, BIRC5, TP53, and MKI67, similar to that reported in biopsy specimens of pulmonary epithelium described to be preneoplastic lesions.

  16. Microfluidic Cell Culture Device

    Science.gov (United States)

    Takayama, Shuichi (Inventor); Cabrera, Lourdes Marcella (Inventor); Heo, Yun Seok (Inventor); Smith, Gary Daniel (Inventor)

    2014-01-01

    Microfluidic devices for cell culturing and methods for using the same are disclosed. One device includes a substrate and membrane. The substrate includes a reservoir in fluid communication with a passage. A bio-compatible fluid may be added to the reservoir and passage. The reservoir is configured to receive and retain at least a portion of a cell mass. The membrane acts as a barrier to evaporation of the bio-compatible fluid from the passage. A cover fluid may be added to cover the bio-compatible fluid to prevent evaporation of the bio-compatible fluid.

  17. Perfusion based cell culture chips

    DEFF Research Database (Denmark)

    Heiskanen, Arto; Emnéus, Jenny; Dufva, Martin

    2010-01-01

    Performing cell culture in miniaturized perfusion chambers gives possibilities to experiment with cells under near in vivo like conditions. In contrast to traditional batch cultures, miniaturized perfusion systems provide precise control of medium composition, long term unattended cultures...... and tissue like structuring of the cultures. However, as this chapter illustrates, many issues remain to be identified regarding perfusion cell culture such as design, material choice and how to use these systems before they will be widespread amongst biomedical researchers....

  18. A Culture-Behavior-Brain Loop Model of Human Development.

    Science.gov (United States)

    Han, Shihui; Ma, Yina

    2015-11-01

    Increasing evidence suggests that cultural influences on brain activity are associated with multiple cognitive and affective processes. These findings prompt an integrative framework to account for dynamic interactions between culture, behavior, and the brain. We put forward a culture-behavior-brain (CBB) loop model of human development that proposes that culture shapes the brain by contextualizing behavior, and the brain fits and modifies culture via behavioral influences. Genes provide a fundamental basis for, and interact with, the CBB loop at both individual and population levels. The CBB loop model advances our understanding of the dynamic relationships between culture, behavior, and the brain, which are crucial for human phylogeny and ontogeny. Future brain changes due to cultural influences are discussed based on the CBB loop model. Copyright © 2015 Elsevier Ltd. All rights reserved.

  19. Reprogramming Cells for Brain Repair

    Directory of Open Access Journals (Sweden)

    Randall D. McKinnon

    2013-08-01

    Full Text Available At present there are no clinical therapies that can repair traumatic brain injury, spinal cord injury or degenerative brain disease. While redundancy and rewiring of surviving circuits can recover some lost function, the brain and spinal column lack sufficient endogenous stem cells to replace lost neurons or their supporting glia. In contrast, pre-clinical studies have demonstrated that exogenous transplants can have remarkable efficacy for brain repair in animal models. Mesenchymal stromal cells (MSCs can provide paracrine factors that repair damage caused by ischemic injury, and oligodendrocyte progenitor cell (OPC grafts give dramatic functional recovery from spinal cord injury. These studies have progressed to clinical trials, including human embryonic stem cell (hESC-derived OPCs for spinal cord repair. However, ESC-derived allografts are less than optimal, and we need to identify a more appropriate donor graft population. The cell reprogramming field has developed the ability to trans-differentiate somatic cells into distinct cell types, a technology that has the potential to generate autologous neurons and glia which address the histocompatibility concerns of allografts and the tumorigenicity concerns of ESC-derived grafts. Further clarifying how cell reprogramming works may lead to more efficient direct reprogram approaches, and possibly in vivo reprogramming, in order to promote brain and spinal cord repair.

  20. Cholera toxin regulates a signaling pathway critical for the expansion of neural stem cell cultures from the fetal and adult rodent brains.

    Directory of Open Access Journals (Sweden)

    Andreas Androutsellis-Theotokis

    2010-05-01

    Full Text Available New mechanisms that regulate neural stem cell (NSC expansion will contribute to improved assay systems and the emerging regenerative approach that targets endogenous stem cells. Expanding knowledge on the control of stem cell self renewal will also lead to new approaches for targeting the stem cell population of cancers.Here we show that Cholera toxin regulates two recently characterized NSC markers, the Tie2 receptor and the transcription factor Hes3, and promotes the expansion of NSCs in culture. Cholera toxin increases immunoreactivity for the Tie2 receptor and rapidly induces the nuclear localization of Hes3. This is followed by powerful cultured NSC expansion and induction of proliferation both in the presence and absence of mitogen.Our data suggest a new cell biological mechanism that regulates the self renewal and differentiation properties of stem cells, providing a new logic to manipulate NSCs in the context of regenerative disease and cancer.

  1. Rescue of Brain Function Using Tunneling Nanotubes Between Neural Stem Cells and Brain Microvascular Endothelial Cells.

    Science.gov (United States)

    Wang, Xiaoqing; Yu, Xiaowen; Xie, Chong; Tan, Zijian; Tian, Qi; Zhu, Desheng; Liu, Mingyuan; Guan, Yangtai

    2016-05-01

    Evidence indicates that neural stem cells (NSCs) can ameliorate cerebral ischemia in animal models. In this study, we investigated the mechanism underlying one of the neuroprotective effects of NSCs: tunneling nanotube (TNT) formation. We addressed whether the control of cell-to-cell communication processes between NSCs and brain microvascular endothelial cells (BMECs) and, particularly, the control of TNT formation could influence the rescue function of stem cells. In an attempt to mimic the cellular microenvironment in vitro, a co-culture system consisting of terminally differentiated BMECs from mice in a distressed state and NSCs was constructed. Additionally, engraftment experiments with infarcted mouse brains revealed that control of TNT formation influenced the effects of stem cell transplantation in vivo. In conclusion, our findings provide the first evidence that TNTs exist between NSCs and BMECs and that regulation of TNT formation alters cell function.

  2. Impaired cholesterol esterification in primary brain cultures of the lysosomal cholesterol storage disorder (LCSD) mouse mutant

    International Nuclear Information System (INIS)

    Patel, S.C.; Suresh, S.; Weintroub, H.; Brady, R.O.; Pentchev, P.G.

    1987-01-01

    Esterification of cholesterol was investigated in primary neuroglial cultures obtained from newborn lysosomal cholesterol storage disorder (LCSD) mouse mutants. An impairment in 3 H-oleic acid incorporation into cholesteryl esters was demonstrated in cultures of homozygous LCSD brain. Primary cultures derived from other phenotypically normal pups of the carrier breeders esterified cholesterol at normal levels or at levels which were intermediary between normal and deficient indicating a phenotypic expression of the LCSD heterozygote genotype. These observations on LCSD mutant brain cells indicate that the defect in cholesterol esterification is closely related to the primary genetic defect and is expressed in neuroglial cells in culture

  3. Ureaplasma infection of cell cultures.

    Science.gov (United States)

    Kotani, H; McGarrity, G J

    1986-05-01

    Studies were performed to characterize the effects of ureaplasmas in HeLa, 3T6, and CV-1 cell cultures. The ureaplasmas studied were human Ureaplasma urealyticum T960 (serotype VIII), bovine U. diversum T95, simian strain T167-2, ovine strain 1202, canine strain D1M-C, and feline strains 382 and FT2-B. FT2-B was the only ureaplasma to grow in the cell free culture medium, Dulbecco modified Eagle-Earle medium containing 10% fetal bovine serum. The growth pattern of the ureaplasmas varied in the different cell cultures, but each strain grew in at least two of the cell cultures, suggesting a requirement for a product of the cell culture and for low concentrations of urea. When growth occurred, organisms grew to concentrations that approached, but did not equal, those observed in 10B broth. Most, but not all, ureaplasmas grew quickly, reaching peak titers 2 days after infection. Canine strain D1M-C did not grow in 3T6, but showed rapid growth in HeLa and CV-1 cells, killing both cultures, In some systems, e.g., U. urealyticum T960 and simian strain T167-2, the infection persisted, and ureaplasmas could be recovered from cell cultures four passages after infection, when studies were terminated. The cell culture ureaplasmas grew on T agar, but not on mycoplasma agar medium.

  4. Endothelial cell marker PAL-E reactivity in brain tumor, developing brain, and brain disease

    NARCIS (Netherlands)

    Leenstra, S.; Troost, D.; Das, P. K.; Claessen, N.; Becker, A. E.; Bosch, D. A.

    1993-01-01

    The endothelial cell marker PAL-E is not reactive to vessels in the normal brain. The present study concerns the PAL-E reactivity in brain tumors in contrast to normal brain and nonneoplastic brain disease. A total of 122 specimens were examined: brain tumors (n = 94), nonneoplastic brain disease (n

  5. Transferrin receptor expression and role in transendothelial transport of transferrin in cultured brain endothelial monolayers

    DEFF Research Database (Denmark)

    Hersom, Maria; Helms, Hans Christian; Pretzer, Natasia

    2016-01-01

    across the endothelial cells by transcytosis. The aim of the present study was to investigate transferrin receptor expression and role in transendothelial transferrin transport in cultured bovine brain endothelial cell monolayers. Transferrin receptor mRNA and protein levels were investigated......Receptor-mediated transcytosis of the transferrin receptor has been suggested as a potential transport system to deliver therapeutic molecules into the brain. Recent studies have however shown that therapeutic antibodies, which have been reported to cross the brain endothelium, reach greater brain...... in endothelial mono-cultures and co-cultures with astrocytes, as well as in freshly isolated brain capillaries using qPCR, immunocytochemistry and Western blotting. Transendothelial transport and luminal association of holo-transferrin was investigated using [125I]holo-transferrin or [59Fe...

  6. Brain tumor stem cell dancing.

    Science.gov (United States)

    Bozzuto, Giuseppina; Toccacieli, Laura; Mazzoleni, Stefania; Frustagli, Gianluca; Chistolini, Pietro; Galli, Rossella; Molinari, Agnese

    2014-01-01

    Issues regarding cancer stem cell (CSC) movement are important in neurosphere biology as cell-cell or cell-environment interactions may have significant impacts on CSC differentiation and contribute to the heterogeneity of the neurosphere. Despite the growing body of literature data on the biology of brain tumor stem cells, floating CSC-derived neurospheres have been scarcely characterized from a morphological and ultrastructural point of view. Here we report a morphological and ultrastructural characterization performed by live imaging and scanning electron microscopy. Glioblastoma multiforme (GBM) CSC-derived neurospheres are heterogeneous and are constituted by cells, morphologically different, capable of forming highly dynamic structures. These dynamic structures are regulated by not serendipitous cell-cell interactions, and they synchronously pulsate following a cyclic course made of "fast" and "slow" alternate phases. Autocrine/paracrine non canonical Wnt signalling appears to be correlated with the association status of neurospheres. The results obtained suggest that GBM CSCs can behave both as independents cells and as "social" cells, highly interactive with other members of its species, giving rise to a sort of "multicellular organism".

  7. Brain tissue banking for stem cells for our future.

    Science.gov (United States)

    Palmero, Emily; Palmero, Sheryl; Murrell, Wayne

    2016-12-19

    In our lab we study neurogenesis and the development of brain tumors. We work towards treatment strategies for glioblastoma and towards using autologous neural stem cells for tissue regeneration strategies for brain damage and neurodegenerative disorders. It has been our policy to try to establish living cell cultures from all human biopsy material that we obtain. We hypothesized that small pieces of brain tissue could be cryopreserved and that live neural stem cells could be recovered at a later time. DMSO has been shown to possess a remarkable ability to diffuse through cell membranes and pass into cell interiors. Its chemical properties prevent the formation of damaging ice crystals thus allowing cell storage at or below -180 C. We report here a protocol for successful freezing of small pieces of tissue derived from human brain and human brain tumours. Virtually all specimens could be successfully revived. Assays of phenotype and behaviour show that the cell cultures derived were equivalent to those cultures previously derived from fresh tissue.

  8. Brain repair: cell therapy in stroke

    Directory of Open Access Journals (Sweden)

    Kalladka D

    2014-02-01

    Full Text Available Dheeraj Kalladka, Keith W Muir Institute of Neuroscience and Psychology, University of Glasgow, Southern General Hospital, Glasgow, United Kingdom Abstract: Stroke affects one in every six people worldwide, and is the leading cause of adult disability. Some spontaneous recovery is usual but of limited extent, and the mechanisms of late recovery are not completely understood. Endogenous neurogenesis in humans is thought to contribute to repair, but its extent is unknown. Exogenous cell therapy is promising as a means of augmenting brain repair, with evidence in animal stroke models of cell migration, survival, and differentiation, enhanced endogenous angiogenesis and neurogenesis, immunomodulation, and the secretion of trophic factors by stem cells from a variety of sources, but the potential mechanisms of action are incompletely understood. In the animal models of stroke, both mesenchymal stem cells (MSCs and neural stem cells (NSCs improve functional recovery, and MSCs reduce the infarct volume when administered acutely, but the heterogeneity in the choice of assessment scales, publication bias, and the possible confounding effects of immunosuppressants make the comparison of effects across cell types difficult. The use of adult-derived cells avoids the ethical issues around embryonic cells but may have more restricted differentiation potential. The use of autologous cells avoids rejection risk, but the sources are restricted, and culture expansion may be necessary, delaying treatment. Allogeneic cells offer controlled cell numbers and immediate availability, which may have advantages for acute treatment. Early clinical trials of both NSCs and MSCs are ongoing, and clinical safety data are emerging from limited numbers of selected patients. Ongoing research to identify prognostic imaging markers may help to improve patient selection, and the novel imaging techniques may identify biomarkers of recovery and the mechanism of action for cell

  9. Mutation in cultured mammalian cells

    International Nuclear Information System (INIS)

    Nakamura, N.; Okada, S.

    1982-01-01

    Mammalian cell cultures were exposed to gamma-rays at various dose rates. Dose-rate effects were observed in cultured somatic cells of the mouse for cell killing and mutations resistant to 6-thioguanine (TGsup(r)) and to methotrexate (MTXsup(r)). Linear quadratic model may be applied to cell killing and TGsup(r) mutations in some cases but can not explain the whole data. Results at low doses with far low dose-rate were not predictable from data at high doses with acute or chronic irradiation. Radioprotective effects of dimethyl sulfoxide were seen only after acute exposure but not after chronic one, suggesting that damages by indirect action of radiations may be potentially reparable by cells. TGsup(r) mutations seem to contain gross structural changes whereas MTXsup(r) ones may have smaller alterations. (Namekawa, K.)

  10. Cell culture compositions

    Science.gov (United States)

    Dunn-Coleman, Nigel; Goedegebuur, Frits; Ward, Michael; Yiao, Jian

    2014-03-18

    The present invention provides a novel endoglucanase nucleic acid sequence, designated egl6 (SEQ ID NO:1 encodes the full length endoglucanase; SEQ ID NO:4 encodes the mature form), and the corresponding endoglucanase VI amino acid sequence ("EGVI"; SEQ ID NO:3 is the signal sequence; SEQ ID NO:2 is the mature sequence). The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding EGVI, recombinant EGVI proteins and methods for producing the same.

  11. Training stem cells for treatment of malignant brain tumors

    Science.gov (United States)

    Li, Shengwen Calvin; Kabeer, Mustafa H; Vu, Long T; Keschrumrus, Vic; Yin, Hong Zhen; Dethlefs, Brent A; Zhong, Jiang F; Weiss, John H; Loudon, William G

    2014-01-01

    The treatment of malignant brain tumors remains a challenge. Stem cell technology has been applied in the treatment of brain tumors largely because of the ability of some stem cells to infiltrate into regions within the brain where tumor cells migrate as shown in preclinical studies. However, not all of these efforts can translate in the effective treatment that improves the quality of life for patients. Here, we perform a literature review to identify the problems in the field. Given the lack of efficacy of most stem cell-based agents used in the treatment of malignant brain tumors, we found that stem cell distribution (i.e., only a fraction of stem cells applied capable of targeting tumors) are among the limiting factors. We provide guidelines for potential improvements in stem cell distribution. Specifically, we use an engineered tissue graft platform that replicates the in vivo microenvironment, and provide our data to validate that this culture platform is viable for producing stem cells that have better stem cell distribution than with the Petri dish culture system. PMID:25258664

  12. The social and cultural roots of whale and dolphin brains.

    Science.gov (United States)

    Fox, Kieran C R; Muthukrishna, Michael; Shultz, Susanne

    2017-11-01

    Encephalization, or brain expansion, underpins humans' sophisticated social cognition, including language, joint attention, shared goals, teaching, consensus decision-making and empathy. These abilities promote and stabilize cooperative social interactions, and have allowed us to create a 'cognitive' or 'cultural' niche and colonize almost every terrestrial ecosystem. Cetaceans (whales and dolphins) also have exceptionally large and anatomically sophisticated brains. Here, by evaluating a comprehensive database of brain size, social structures and cultural behaviours across cetacean species, we ask whether cetacean brains are similarly associated with a marine cultural niche. We show that cetacean encephalization is predicted by both social structure and by a quadratic relationship with group size. Moreover, brain size predicts the breadth of social and cultural behaviours, as well as ecological factors (diversity of prey types and to a lesser extent latitudinal range). The apparent coevolution of brains, social structure and behavioural richness of marine mammals provides a unique and striking parallel to the large brains and hyper-sociality of humans and other primates. Our results suggest that cetacean social cognition might similarly have arisen to provide the capacity to learn and use a diverse set of behavioural strategies in response to the challenges of social living.

  13. Culture and attention: evidence from brain and behavior.

    Science.gov (United States)

    Ketay, Sarah; Aron, Arthur; Hedden, Trey

    2009-01-01

    Research has demonstrated that our experiences, including the culture in which we are raised, shape how we attend to and perceive the world. Behavioral studies have found that individuals raised in Western cultures tend toward analytic processing and prefer tasks emphasizing independent contexts rather than tasks emphasizing interdependent contexts. The opposite is true for individuals raised in East Asian cultures, who tend toward holistic processing and prefer tasks emphasizing interdependent contexts. Recently, cognitive neuroscientists have extended these behavioral findings to examine the brain activity of individuals from different cultures during the performance of cognitive tasks. Results from these initial studies indicate that culture may shape how the brain processes even very abstract stimuli and may influence the features of the environment to which individuals attend. The present chapter reviews evidence that culture influences attention and related systems, which, in turn, impact other cognitive and social processes and their neural correlates.

  14. Development and Characterization of a Brain Endothelial Cell Phenotype using Human Induced Pluripotent Stem Cells

    DEFF Research Database (Denmark)

    Goldeman, Charlotte; Saaby, Lasse; Holst, Bjørn

    The transport of substances from blood to brain is regulated by the blood-brain barrier (BBB), i.e. the barrier properties of the brain endothelium. The endothelium restricts the transport into the brain of the majority of new drug candidates. Cultured monolayers of brain endothelial cells can...... be used to investigate drug transport in vitro, and screen candidates for permeation properties. One recent approach is to develop in vitro models of the BBB using human induced pluripotent stem cells (hIPSCs) as described by Stebbins et al. (2015).The aim of the present study was to investigate whether...... the published protocols were generically applicable and thus to develop and characterize in vitro models of the BBB using hIPSCs from different sources. Two stem cell lines, Bioni010-C and WTSli024-A, were seeded and maintained on Matrigel in mTesR1 media. Cells were then seeded as single cells at different...

  15. Youth Culture and Cell Phone

    Directory of Open Access Journals (Sweden)

    mohammad saeed zokaei

    2009-11-01

    Full Text Available Iranian youth’s leisure culture has been immediately affected by the digital media culture. As a communicative media, cell phone has crossed borders of youth norms and identity; and in addition to facilitating their communication, has changed its patterns. Applying Bourdieu’s concepts of habitus and field, and relied on the qualitative and quantitative data gathered from the mobile youth users, the present study argues that mobile has produced a new field in which youth’s opportunities for leisure, entertainment, communication, and independence have extended. In addition, cell phone has facilitated and compensated for some defects in public sphere, and therefore empowered youth agency, individuality, and power. Despite this strengthening, cell phone does not cross borders of gender and class differences, or the levels of social capital.

  16. Biomimetic brain tumor niche regulates glioblastoma cells towards a cancer stem cell phenotype.

    Science.gov (United States)

    Liu, Yung-Chiang; Lee, I-Chi; Chen, Pin-Yuan

    2018-05-01

    Glioblastoma (GBM) is the most malignant primary brain tumor and contains tumorigenic cancer stem cells (CSCs), which support the progression of tumor growth. The selection of CSCs and facilitation of the brain tumor niches may assist the development of novel therapeutics for GBM. Herein, hydrogel materials composed of agarose and hydroxypropyl methyl cellulose (HMC) in different concentrations were established and compared to emulate brain tumor niches and CSC microenvironments within a label-free system. Human GBM cell line, U-87 MG, was cultured on a series of HMC-agarose based culture system. Cell aggregation and spheroids formation were investigated after 4 days of culture, and 2.5% HMC-agarose based culture system demonstrated the largest spheroids number and size. Moreover, CD133 marker expression of GBM cells after 6 days of culture in 2.5% HMC-agarose based culture system was 60%, relatively higher than the control group at only 15%. Additionally, cells on 2.5% HMC-agarose based culture system show the highest chemoresistance, even at the high dose of 500 µM temozolomide for 72 h, the live cell ratio was still > 80%. Furthermore, the results also indicate that the expression of ABCG2 gene was up-regulated after culture in 2.5% HMC-agarose based culture system. Therefore, our results demonstrated that biomimetic brain tumor microenvironment may regulate GBM cells towards the CSC phenotype and expression of CSC characteristics. The microenvironment selection and spheroids formation in HMC-agarose based culture system may provide a label-free CSC selection strategy and drug testing model for future biomedical applications.

  17. Xanthine oxidase activity regulates human embryonic brain cells growth

    Directory of Open Access Journals (Sweden)

    Kevorkian G. A.

    2011-10-01

    Full Text Available Aim. Involvement of Xanthine Oxidase (XO; EC1.1.3.22 in cellular proliferation and differentiation has been suggested by the numerous investigations. We have proposed that XO might have undoubtedly important role during the development, maturation as well as the death of human embryos brain cells. Methods. Human abortion material was utilized for the cultivation of brain cells (E90. XO activity was measured by the formation of uric acid in tissue. Cell death was detected by the utility of Trypan Blue dye. Results. Allopurinol suppressed the XO activity in the brain tissue (0.12 ± 0.02; 0.20 ± 0.03 resp., p < 0.05. On day 12th the number of cells in the culture treated with the Allopurinol at the early stage of development was higher in comparison with the Control (2350.1 ± 199.0 vs 2123 ± 96 and higher in comparison with the late period of treatment (1479.6 ± 103.8, p < < 0.05. In all groups, the number of the dead cells was less than in Control, indicating the protective nature of Allopurinol as an inhibitor of XO. Conclusions. Allopurinol initiates cells proliferation in case of the early treatment of the human brain derived cell culture whereas at the late stages it has an opposite effect.

  18. Barrier Functionality of Porcine and Bovine Brain Capillary Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Ailar Nakhlband

    2011-09-01

    Full Text Available Introduction: To date, isolated cell based blood-brain barrier (BBB models have been widely used for brain drug delivery and targeting, due to their relatively proper bioelectrical and permeability properties. However, primary cultures of brain capillary endothelial cells (BCECs isolated from different species vary in terms of bioelectrical and permeability properties. Methods: To pursue this, in the current investigation, primary porcine and bovine BCECs (PBCECs and BBCECs, respectively were isolated and used as an in vitro BBB model. The bioelectrical and permeability properties were assessed in BCECs co-cultured with C6 cells with/without hydrocortisone (550 nM. The bioelectrical properties were further validated by means of the permeability coefficients of transcellular and paracellular markers. Results: The primary PBCECs displayed significantly higher trans-endothelial electrical resistance (~900 W.cm2 than BBCECs (~700 W.cm2 - both co-cultured with C6 cells in presence of hydrocortisone. Permeability coefficients of propranolol/diazepam and mannitol/sucrose in PBCECs were ~21 and ~2 (×10-6 cm.sec-1, where these values for BBCECs were ~25 and ~5 (×10-6 cm.sec-1. Conclusion: Upon our bioelectrical and permeability findings, both models display discriminative barrier functionality but porcine BCECs seem to provide a better platform than bovine BCECs for drug screening and brain targeting.

  19. Permeability of PEGylated immunoarsonoliposomes through in vitro blood brain barrier-medulloblastoma co-culture models for brain tumor therapy.

    Science.gov (United States)

    Al-Shehri, Abdulghani; Favretto, Marco E; Ioannou, Panayiotis V; Romero, Ignacio A; Couraud, Pierre-Olivier; Weksler, Babette Barbash; Parker, Terry L; Kallinteri, Paraskevi

    2015-03-01

    Owing to restricted access of pharmacological agents into the brain due to blood brain barrier (BBB) there is a need: 1. to develop a more representative 3-D-co-culture model of tumor-BBB interaction to investigate drug and nanoparticle transport into the brain for diagnostic and therapeutic evaluation. 2. to address the lack of new alternative methods to animal testing according to replacement-reduction-refinement principles. In this work, in vitro BBB-medulloblastoma 3-D-co-culture models were established using immortalized human primary brain endothelial cells (hCMEC/D3). hCMEC/D3 cells were cultured in presence and in absence of two human medulloblastoma cell lines on Transwell membranes. In vitro models were characterized for BBB formation, zonula occludens-1 expression and permeability to dextran. Transferrin receptors (Tfr) expressed on hCMEC/D3 were exploited to facilitate arsonoliposome (ARL) permeability through the BBB to the tumor by covalently attaching an antibody specific to human Tfr. The effect of anticancer ARLs on hCMEC/D3 was assessed. In vitro BBB and BBB-tumor co-culture models were established successfully. BBB permeability was affected by the presence of tumor aggregates as suggested by increased permeability of ARLs. There was a 6-fold and 8-fold increase in anti-Tfr-ARL uptake into VC312R and BBB-DAOY co-culture models, respectively, compared to plain ARLs. The three-dimensional models might be appropriate models to study the transport of various drugs and nanocarriers (liposomes and immunoarsonoliposomes) through the healthy and diseased BBB. The immunoarsonoliposomes can be potentially used as anticancer agents due to good tolerance of the in vitro BBB model to their toxic effect.

  20. Issues of cultural diversity in acquired brain injury (ABI) rehabilitation.

    Science.gov (United States)

    Lequerica, Anthony; Krch, Denise

    2014-01-01

    With the general population in the United States becoming increasingly diverse, it is important for rehabilitation professionals to develop the capacity to provide culturally sensitive treatment. This is especially relevant when working with minority populations who have a higher risk for brain injury and poorer rehabilitation outcomes. This article presents a number of clinical vignettes to illustrate how cultural factors can influence behavior in patients recovering from brain injury, as well as rehabilitation staff. The main objectives are to raise awareness among clinicians and stimulate research ideas by highlighting some real world examples of situations where a specialized, patient-centered approach needs to consider factors of cultural diversity. Because one's own world view impacts the way we see the world and interpret behavior, it is important to understand one's own ethnocentrism when dealing with a diverse population of patients with brain injury where behavioral sequelae are often expected. Being able to see behavior after brain injury with an open mind and taking into account cultural and contextual factors is an important step in developing culturally competent rehabilitation practices.

  1. Basic Techniques in Mammalian Cell Tissue Culture.

    Science.gov (United States)

    Phelan, Katy; May, Kristin M

    2016-11-01

    Cultured mammalian cells are used extensively in cell biology studies. It requires a number of special skills in order to be able to preserve the structure, function, behavior, and biology of the cells in culture. This unit describes the basic skills required to maintain and preserve cell cultures: maintaining aseptic technique, preparing media with the appropriate characteristics, passaging, freezing and storage, recovering frozen stocks, and counting viable cells. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

  2. Stem cells to regenerate the newborn brain

    NARCIS (Netherlands)

    van Velthoven, C.T.J.

    2011-01-01

    Perinatal hypoxia-ischemia (HI) is a frequent cause of perinatal morbidity and mortality with limited therapeutic options. In this thesis we investigate whether mesenchymal stem cells (MSC) regenerate the neonatal brain after HI injury. We show that transplantation of MSC after neonatal brain injury

  3. New Nerve Cells for the Adult Brain.

    Science.gov (United States)

    Kempermann, Gerd; Gage, Fred H.

    1999-01-01

    Contrary to dogma, the human brain does produce new nerve cells in adulthood. The mature human brain spawns neurons routinely in the hippocampus, an area important to memory and learning. This research can make it possible to ease any number of disorders involving neurological damage and death. (CCM)

  4. CD90 Expression on human primary cells and elimination of contaminating fibroblasts from cell cultures.

    Science.gov (United States)

    Kisselbach, Lynn; Merges, Michael; Bossie, Alexis; Boyd, Ann

    2009-01-01

    Cluster Differentiation 90 (CD90) is a cell surface glycoprotein originally identified on mouse thymocytes. Although CD90 has been identified on a variety of stem cells and at varying levels in non-lymphoid tissues such as on fibroblasts, brain cells, and activated endothelial cells, the knowledge about the levels of CD90 expression on different cell types, including human primary cells, is limited. The goal of this study was to identify CD90 as a human primary cell biomarker and to develop an efficient and reliable method for eliminating unwanted or contaminating fibroblasts from human primary cell cultures suitable for research pursuant to cell based therapy technologies.

  5. Radiation Response of Cultured Human Cells Is Unaffected by Johrei

    OpenAIRE

    Hall, Zach; Luu, Tri; Moore, Dan; Yount, Garret

    2007-01-01

    Johrei has been credited with healing thousands from radiation wounds after the Hiroshima and Nagasaki bombs in 1945. This alternative medical therapy is becoming increasingly popular in the United States, as are other Energy Medicine modalities that purport to influence a universal healing energy. Human brain cells were cultured and exposed to increasing doses of ionizing radiation. Experienced Johrei practitioners directed healing intentionality toward the cells for 30 min from a distance o...

  6. 9 CFR 101.6 - Cell cultures.

    Science.gov (United States)

    2010-01-01

    ... used in conjunction with or in reference to cell cultures, which may be referred to as tissue cultures... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Cell cultures. 101.6 Section 101.6 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES...

  7. Uptake of dimercaptosuccinate-coated magnetic iron oxide nanoparticles by cultured brain astrocytes

    International Nuclear Information System (INIS)

    Geppert, Mark; Hohnholt, Michaela C; Dringen, Ralf; Thiel, Karsten; Grunwald, Ingo; Nuernberger, Sylvia; Rezwan, Kurosch

    2011-01-01

    Magnetic iron oxide nanoparticles (Fe-NP) are currently considered for various diagnostic and therapeutic applications in the brain. However, little is known on the accumulation and biocompatibility of such particles in brain cells. We have synthesized and characterized dimercaptosuccinic acid (DMSA) coated Fe-NP and have investigated their uptake by cultured brain astrocytes. DMSA-coated Fe-NP that were dispersed in physiological medium had an average hydrodynamic diameter of about 60 nm. Incubation of cultured astrocytes with these Fe-NP caused a time- and concentration-dependent accumulation of cellular iron, but did not lead within 6 h to any cell toxicity. After 4 h of incubation with 100-4000 μM iron supplied as Fe-NP, the cellular iron content reached levels between 200 and 2000 nmol mg -1 protein. The cellular iron content after exposure of astrocytes to Fe-NP at 4 deg. C was drastically lowered compared to cells that had been incubated at 37 deg. C. Electron microscopy revealed the presence of Fe-NP-containing vesicles in cells that were incubated with Fe-NP at 37 deg. C, but not in cells exposed to the nanoparticles at 4 deg. C. These data demonstrate that cultured astrocytes efficiently take up DMSA-coated Fe-NP in a process that appears to be saturable and strongly depends on the incubation temperature.

  8. Comparative Analysis of Human and Rodent Brain Primary Neuronal Culture Spontaneous Activity Using Micro-Electrode Array Technology.

    Science.gov (United States)

    Napoli, Alessandro; Obeid, Iyad

    2016-03-01

    Electrical activity in embryonic brain tissue has typically been studied using Micro Electrode Array (MEA) technology to make dozens of simultaneous recordings from dissociated neuronal cultures, brain stem cell progenitors, or brain slices from fetal rodents. Although these rodent neuronal primary culture electrical properties are mostly investigated, it has not been yet established to what extent the electrical characteristics of rodent brain neuronal cultures can be generalized to those of humans. A direct comparison of spontaneous spiking activity between rodent and human primary neurons grown under the same in vitro conditions using MEA technology has never been carried out before and will be described in the present study. Human and rodent dissociated fetal brain neuronal cultures were established in-vitro by culturing on a glass grid of 60 planar microelectrodes neurons under identical conditions. Three different cultures of human neurons were produced from tissue sourced from a single aborted fetus (at 16-18 gestational weeks) and these were compared with seven different cultures of embryonic rat neurons (at 18 gestational days) originally isolated from a single rat. The results show that the human and rodent cultures behaved significantly differently. Whereas the rodent cultures demonstrated robust spontaneous activation and network activity after only 10 days, the human cultures required nearly 40 days to achieve a substantially weaker level of electrical function. These results suggest that rat neuron preparations may yield inferences that do not necessarily transfer to humans. © 2015 Wiley Periodicals, Inc.

  9. Islam, brain death, and transplantation: culture, faith, and jurisprudence.

    Science.gov (United States)

    Arbour, Richard; AlGhamdi, Hanan Mesfer Saad; Peters, Linda

    2012-01-01

    A significant gap exists between availability of organs for transplant and patients with end-stage organ failure for whom organ transplantation is the last treatment option. Reasons for this mismatch include inadequate approach to potential donor families and donor loss as a result of refractory cardiopulmonary instability during and after brainstem herniation. Other reasons include inadequate cultural competence and sensitivity when communicating with potential donor families. Clinicians may not have an understanding of the cultural and religious perspectives of Muslim families of critically ill patients who may be approached about brain death and organ donation. This review analyzes Islamic cultural and religious perspectives on organ donation, transplantation, and brain death, including faith-based directives from Islamic religious authorities, definitions of death in Islam, and communication strategies when discussing brain death and organ donation with Muslim families. Optimal family care and communication are highlighted using case studies and backgrounds illustrating barriers and approaches with Muslim families in the United States and in the Kingdom of Saudi Arabia that can improve cultural competence and family care as well as increase organ availability within the Muslim population and beyond.

  10. Comparison of primary skunk brain and kidney and raccoon kidney cells with established cell lines for isolation and propagation of street rabies virus.

    OpenAIRE

    Umoh, J U; Blenden, D C

    1983-01-01

    Cell cultures prepared from skunk kidney, raccoon kidney, and skunk brain were compared with CER, murine neuroblastoma (C1300, clone NA), baby hamster kidney (BHK-21, S-13), and dog kidney (MDCK) cell lines for virus isolation and propagation of street and fixed rabies virus. The skunk brain cells were suitable for efficient replication of all the virus isolates. They were comparable to CER and murine neuroblastoma cells for virus isolation and propagation. None of the other cell cultures was...

  11. [Isolation and identification of brain tumor stem cells from human brain neuroepithelial tumors].

    Science.gov (United States)

    Fang, Jia-sheng; Deng, Yong-wen; Li, Ming-chu; Chen, Feng-Hua; Wang, Yan-jin; Lu, Ming; Fang, Fang; Wu, Jun; Yang, Zhuan-yi; Zhou, Xang-yang; Wang, Fei; Chen, Cheng

    2007-01-30

    To establish a simplified culture system for the isolation of brain tumor stem cells (BTSCs) from the tumors of human neuroepithelial tissue, to observe the growth and differentiation pattern of BTSCs, and to investigate their expression of the specific markers. Twenty-six patients with brain neuroepithelial tumors underwent tumor resection. Two pieces of tumor tissues were taken from each tumor to be dissociated, triturated into single cells in sterile DMEM-F12 medium, and then filtered. The tumor cells were seeded at a concentration of 200,000 viable cells per mL into serum-free DMEM-F12 medium simply supplemented with B27, human basic fibroblast growth factor (20 microg/L), human epidermal growth factor (20 microg /L), insulin (4 U/L), L-glutamine, penicillin and streptomycin. After the primary brain tumor spheres (BTSs) were generated, they were triturated again and passed in fresh medium. Limiting dilution assay was performed to observe the monoclone formation. 5-bromodeoxyuridine (BrdU) incorporation test was performed to observe the proliferation of the BTS. The BTSCs were cultured in mitogen-free DMEM-F12 medium supplemented with 10% fetal bovine serum to observe their differentiation. Immunocytochemistry was used to examine the expression of CD133 and nestin, specific markers of BTSC, and the rate of CD133 positive cells. Only a minority of subsets of cells from the tumors of neuroepithelial tissue had the capacity to survive, proliferate, and generate free-floating neurosphere-like BTSs in the simplified serum-free medium. These cells attached to the poly-L-lysine coated coverslips in the serum-supplemented medium and differentiated. The BTSCs were CD133 and nestin positive. The rate of CD133 positive cells in the tumor specimens was (21 +/- 6.2)% - (38 +/- 7.0)%. A new simplified culture system for the isolation of BTSCs is established. The tumors of human neuroepithelial tissue contain CD133 and nestin positive tumor stem cells which can be isolated

  12. Topological defects control collective dynamics in neural progenitor cell cultures

    Science.gov (United States)

    Kawaguchi, Kyogo; Kageyama, Ryoichiro; Sano, Masaki

    2017-04-01

    Cultured stem cells have become a standard platform not only for regenerative medicine and developmental biology but also for biophysical studies. Yet, the characterization of cultured stem cells at the level of morphology and of the macroscopic patterns resulting from cell-to-cell interactions remains largely qualitative. Here we report on the collective dynamics of cultured murine neural progenitor cells (NPCs), which are multipotent stem cells that give rise to cells in the central nervous system. At low densities, NPCs moved randomly in an amoeba-like fashion. However, NPCs at high density elongated and aligned their shapes with one another, gliding at relatively high velocities. Although the direction of motion of individual cells reversed stochastically along the axes of alignment, the cells were capable of forming an aligned pattern up to length scales similar to that of the migratory stream observed in the adult brain. The two-dimensional order of alignment within the culture showed a liquid-crystalline pattern containing interspersed topological defects with winding numbers of +1/2 and -1/2 (half-integer due to the nematic feature that arises from the head-tail symmetry of cell-to-cell interaction). We identified rapid cell accumulation at +1/2 defects and the formation of three-dimensional mounds. Imaging at the single-cell level around the defects allowed us to quantify the velocity field and the evolving cell density; cells not only concentrate at +1/2 defects, but also escape from -1/2 defects. We propose a generic mechanism for the instability in cell density around the defects that arises from the interplay between the anisotropic friction and the active force field.

  13. 3D Cell Culture in Alginate Hydrogels

    Directory of Open Access Journals (Sweden)

    Therese Andersen

    2015-03-01

    Full Text Available This review compiles information regarding the use of alginate, and in particular alginate hydrogels, in culturing cells in 3D. Knowledge of alginate chemical structure and functionality are shown to be important parameters in design of alginate-based matrices for cell culture. Gel elasticity as well as hydrogel stability can be impacted by the type of alginate used, its concentration, the choice of gelation technique (ionic or covalent, and divalent cation chosen as the gel inducing ion. The use of peptide-coupled alginate can control cell–matrix interactions. Gelation of alginate with concomitant immobilization of cells can take various forms. Droplets or beads have been utilized since the 1980s for immobilizing cells. Newer matrices such as macroporous scaffolds are now entering the 3D cell culture product market. Finally, delayed gelling, injectable, alginate systems show utility in the translation of in vitro cell culture to in vivo tissue engineering applications. Alginate has a history and a future in 3D cell culture. Historically, cells were encapsulated in alginate droplets cross-linked with calcium for the development of artificial organs. Now, several commercial products based on alginate are being used as 3D cell culture systems that also demonstrate the possibility of replacing or regenerating tissue.

  14. Fluidic system for long-term in vitro culturing and monitoring of organotypic brain slices

    DEFF Research Database (Denmark)

    Bakmand, Tanya; Troels-Smith, Ane R.; Dimaki, Maria

    2015-01-01

    Brain slice preparations cultured in vitro have long been used as a simplified model for studying brain development, electrophysiology, neurodegeneration and neuroprotection. In this paper an open fluidic system developed for improved long term culturing of organotypic brain slices is presented. ...

  15. Isolation and culture of pulmonary endothelial cells.

    OpenAIRE

    Ryan, U S

    1984-01-01

    Methods for isolation, identification and culture of pulmonary endothelial cells are now routine. In the past, methods of isolation have used proteolytic enzymes to detach cells; thereafter, traditional methods for cell passaging have used trypsin/EDTA mixtures. Cells isolated and passaged using proteolytic enzymes have been useful in establishing the field and in verifying certain endothelial properties. However, there is a growing awareness of the role of endothelial cells in processing vas...

  16. Primary brain tumors, neural stem cell, and brain tumor cancer cells: where is the link?

    Science.gov (United States)

    Germano, Isabelle; Swiss, Victoria; Casaccia, Patrizia

    2010-01-01

    The discovery of brain tumor-derived cells (BTSC) with the properties of stem cells has led to the formulation of the hypothesis that neural stem cells could be the cell of origin of primary brain tumors (PBT). In this review we present the most common molecular changes in PBT, define the criteria of identification of BTSC and discuss the similarities between the characteristics of these cells and those of the endogenous population of neural stem cells (NPCs) residing in germinal areas of the adult brain. Finally, we propose possible mechanisms of cancer initiation and progression and suggest a model of tumor initiation that includes intrinsic changes of resident NSC and potential changes in the microenvironment defining the niche where the NSC reside. PMID:20045420

  17. Advances in 3D neuronal cell culture

    NARCIS (Netherlands)

    Frimat, Jean Philippe; Xie, Sijia; Bastiaens, Alex; Schurink, Bart; Wolbers, Floor; Den Toonder, Jaap; Luttge, Regina

    2015-01-01

    In this contribution, the authors present our advances in three-dimensional (3D) neuronal cell culture platform technology contributing to controlled environments for microtissue engineering and analysis of cellular physiological and pathological responses. First, a micromachined silicon sieving

  18. Comparison of prostanoid forming capacity of neuronal and astroglial cells in primary cultures.

    Science.gov (United States)

    Keller, M; Jackisch, R; Seregi, A; Hertting, G

    1985-01-01

    Prostaglandin (PG) and thromboxane (TX) biosynthesis in primary neuronal and astroglial cell cultures was studied. Cultures obtained from fetal (15-16 days old) and neonatal rat brain hemispheres were characterized by chemical and immunocytochemical staining techniques as predominantly neurons or mature and immature astrocytes, respectively. Six-day old neuronal cell cultures grown in the presence of cytosine arabinoside (2 ?M) from the day 3 onwards were contaminated up to 10% with glioblasts. In astroglial cultures up to 3% of the cells were postively stained with a marker for oligodendroglial cells. Fibroblast contamination was below 1% in both cultures. Prostanoid formation (measured by specific radioimmunoassays) in 6-day old neuronal cell cultures was low (sum of the amount of PGs and TX formed: 1.16 +/- 0.17 (ng/mg protein/15 min) as compared to 14-day old cultured astroglial cells: 21.27 +/- 2.53 (ng/mg protein/15 min). Also the pattern of prostanoids formed was different in neuronal (PGD(2) ? PGF(2?) > TXB(2) ? PGE(2)) and astroglial cells (PGD(2) > TXB(2) ? PGF(2?) ? PGE(2) ? 6-ketoPGF(1?)). Preincubation with arachidonic acid (1 ?g/ml) did not affect prostanoid formation in both cultures, whereas it was stimulated 4-6-fold by addition of the calcium ionophore A23187 (1 ?M). These results, although found on cultured neuronal and glial cells of different stages of development, support the view that astroglial cells might play a crucial role in brain prostanoid synthesis.

  19. Social learning, culture and the 'socio-cultural brain' of human and non-human primates.

    Science.gov (United States)

    Whiten, Andrew; van de Waal, Erica

    2017-11-01

    Noting important recent discoveries, we review primate social learning, traditions and culture, together with associated findings about primate brains. We survey our current knowledge of primate cultures in the wild, and complementary experimental diffusion studies testing species' capacity to sustain traditions. We relate this work to theories that seek to explain the enlarged brain size of primates as specializations for social intelligence, that have most recently extended to learning from others and the cultural transmission this permits. We discuss alternative theories and review a variety of recent findings that support cultural intelligence hypotheses for primate encephalization. At a more fine-grained neuroscientific level we focus on the underlying processes of social learning, especially emulation and imitation. Here, our own and others' recent research has established capacities for bodily imitation in both monkeys and apes, results that are consistent with a role for the mirror neuron system in social learning. We review important convergences between behavioural findings and recent non-invasive neuroscientific studies. Copyright © 2016 Elsevier Ltd. All rights reserved.

  20. Excitatory amino acid neurotoxicity and modulation of glutamate receptor expression in organotypic brain slice cultures

    DEFF Research Database (Denmark)

    Zimmer, J; Kristensen, Bjarne Winther; Jakobsen, B

    2000-01-01

    Using organotypic slice cultures of hippocampus and cortex-striatum from newborn to 7 day old rats, we are currently studying the excitotoxic effects of kainic acid (KA), AMPA and NMDA and the neuroprotective effects of glutamate receptor blockers, like NBQX. For detection and quantitation...... of the induced neurodegeneration, we have developed standardized protocols, including--a) densitometric measurements of the cellular uptake of propidium iodide (PI), --b) histological staining by Flouro-Jade, --c) lactate dehydrogenase (LDH) release to the culture medium, --d) immunostaining for microtubulin......-induced excitotoxicity and KA-glutamate receptor subunit mRNA expression after long-term exposure to low, non-toxic doses of KA and NBQX. We conclude that organotypic brain slice cultures, combined with standardized procedures for quantitation of cell damage and receptor subunit changes is of great potential use...

  1. Glial cell ceruloplasmin and hepcidin differentially regulate iron efflux from brain microvascular endothelial cells.

    Science.gov (United States)

    McCarthy, Ryan C; Kosman, Daniel J

    2014-01-01

    We have used an in vitro model system to probe the iron transport pathway across the brain microvascular endothelial cells (BMVEC) of the blood-brain barrier (BBB). This model consists of human BMVEC (hBMVEC) and C6 glioma cells (as an astrocytic cell line) grown in a transwell, a cell culture system commonly used to quantify metabolite flux across a cell-derived barrier. We found that iron efflux from hBMVEC through the ferrous iron permease ferroportin (Fpn) was stimulated by secretion of the soluble form of the multi-copper ferroxidase, ceruloplasmin (sCp) from the co-cultured C6 cells. Reciprocally, expression of sCp mRNA in the C6 cells was increased by neighboring hBMVEC. In addition, data indicate that C6 cell-secreted hepcidin stimulates internalization of hBMVEC Fpn but only when the end-feet projections characteristic of this glia-derived cell line are proximal to the endothelial cells. This hepcidin-dependent loss of Fpn correlated with knock-down of iron efflux from the hBMVEC; this result was consistent with the mechanism by which hepcidin regulates iron efflux in mammalian cells. In summary, the data support a model of iron trafficking across the BBB in which the capillary endothelium induce the underlying astrocytes to produce the ferroxidase activity needed to support Fpn-mediated iron efflux. Reciprocally, astrocyte proximity modulates the effective concentration of hepcidin at the endothelial cell membrane and thus the surface expression of hBMVEC Fpn. These results are independent of the source of hBMVEC iron (transferrin or non-transferrin bound) indicating that the model developed here is broadly applicable to brain iron homeostasis.

  2. Poststroke Cell Therapy of the Aged Brain

    Directory of Open Access Journals (Sweden)

    Aurel Popa-Wagner

    2015-01-01

    Full Text Available During aging, many neurodegenerative disorders are associated with reduced neurogenesis and a decline in the proliferation of stem/progenitor cells. The development of the stem cell (SC, the regenerative therapy field, gained tremendous expectations in the diseases that suffer from the lack of treatment options. Stem cell based therapy is a promising approach to promote neuroregeneration after brain injury and can be potentiated when combined with supportive pharmacological drug treatment, especially in the aged. However, the mechanism of action for a particular grafted cell type, the optimal delivery route, doses, or time window of administration after lesion is still under debate. Today, it is proved that these protections are most likely due to modulatory mechanisms rather than the expected cell replacement. Our group proved that important differences appear in the aged brain compared with young one, that is, the accelerated progression of ischemic area, or the delayed initiation of neurological recovery. In this light, these age-related aspects should be carefully evaluated in the clinical translation of neurorestorative therapies. This review is focused on the current perspectives and suitable sources of stem cells (SCs, mechanisms of action, and the most efficient delivery routes in neurorestoration therapies in the poststroke aged environment.

  3. Isolation and culture of pulmonary endothelial cells.

    Science.gov (United States)

    Ryan, U S

    1984-06-01

    Methods for isolation, identification and culture of pulmonary endothelial cells are now routine. In the past, methods of isolation have used proteolytic enzymes to detach cells; thereafter, traditional methods for cell passaging have used trypsin/EDTA mixtures. Cells isolated and passaged using proteolytic enzymes have been useful in establishing the field and in verifying certain endothelial properties. However, there is a growing awareness of the role of endothelial cells in processing vasoactive substances, in responding to hormones and other agonists and in cell-cell interactions with other cell types of the vascular wall, with blood cells and with cellular products. Consequently, a new requirement has arisen for cells in vitro that maintain the differentiated properties of their counterparts in vivo. The deleterious effects of trypsin and other proteolytic enzymes commonly used in cell culture on surface structures of endothelial cells such as enzymes, receptors and junctional proteins, as well as on extracellular layers such as the glycocalyx or "endothelial fuzz," have led to the development of methods that avoid use of proteolytic enzymes at both the isolation step and during subsequent subculture. This chapter describes traditional methods for isolating pulmonary endothelial cells but emphasizes newer approaches using mechanical harvest and scale-up using microcarriers. The new methods allow maintenance of long-term, large-scale cultures of cells that retain the full complement of surface properties and that maintain the cobblestone monolayer morphology and differentiated functional properties. Methods for identification of isolated cells are therefore also considered as methods for validation of cultures during their in vitro lifespan.

  4. Melphalan metabolism in cultured cells

    International Nuclear Information System (INIS)

    Seagrave, J.C.; Valdez, J.G.; Tobey, R.A.; Gurley, L.R.

    1985-06-01

    Procedures are presented for the adaptation of reversed-phase-HPLC methods to accomplish separation and isolation of the cancer therapeutic drug melphalan (L-phenylalanine mustard) and its metabolic products from whole cells. Five major degradation products of melphalan were observed following its hydrolysis in phosphate buffer in vitro. The two most polar of these products (or modifications of them) were also found in the cytosol of Chinese hamster CHO cells. The amounts of these two polar products (shown not to be mono- or dihydroxymelphalan) were significantly changed by the pretreatment of cells with ZnC1 2 , one being increased in amount while the other was reduced to an insignificant level. In ZnC1 2 -treated cells, there was also an increased binding of melphalan (or its derivatives) to one protein fraction resolved by gel filtration-HPLC. These observations suggest that changes in polar melphalan products, and perhaps their interaction with a protein, may by involved in the reduction of melphalan cytotoxicity observed in ZnC1 2 -treated cells. While ZnC1 2 is also known to increase the level of glutathione in cells, no significant amounts of glutathione-melphalan derivatives of the type formed non-enzymatically in vitro could be detected in ZnC1 2 -treated or untreated cells. Formation of derivatives of melphalan with glutathione catabolic products in ZnC1 2 -treated cells has not yet been eliminated, however. 17 refs., 5 figs., 1 tab

  5. [Stem Cells in the Brain of Mammals and Human: Fundamental and Applied Aspects].

    Science.gov (United States)

    Aleksandrova, M A; Marey, M V

    2015-01-01

    Brain stem cells represent an extremely intriguing phenomenon. The aim of our review is to present an integrity vision of their role in the brain of mammals and humans, and their clinical perspectives. Over last two decades, investigations of biology of the neural stem cells produced significant changes in general knowledge about the processes of development and functioning of the brain. Researches on the cellular and molecular mechanisms of NSC differentiation and behavior led to new understanding of their involvement in learning and memory. In the regenerative medicine, original therapeutic approaches to neurodegenerative brain diseases have been elaborated due to fundamental achievements in this field. They are based on specific regenerative potential of neural stem cells and progenitor cells, which possess the ability to replace dead cells and express crucially significant biologically active factors that are missing in the pathological brain. For the needs of cell substitution therapy in the neural diseases, adequate methods of maintaining stem cells in culture and their differentiation into different types of neurons and glial cells, have been developed currently. The success of modern cellular technologies has significantly expanded the range of cells used for cell therapy. The near future may bring new perspective and distinct progress in brain cell therapy due to optimizing the cells types most promising for medical needs.

  6. Excitatory amino acid neurotoxicity and modulation of glutamate receptor expression in organotypic brain slice cultures

    DEFF Research Database (Denmark)

    Zimmer, J; Kristensen, Bjarne Winther; Jakobsen, B

    2000-01-01

    Using organotypic slice cultures of hippocampus and cortex-striatum from newborn to 7 day old rats, we are currently studying the excitotoxic effects of kainic acid (KA), AMPA and NMDA and the neuroprotective effects of glutamate receptor blockers, like NBQX. For detection and quantitation......-induced excitotoxicity and KA-glutamate receptor subunit mRNA expression after long-term exposure to low, non-toxic doses of KA and NBQX. We conclude that organotypic brain slice cultures, combined with standardized procedures for quantitation of cell damage and receptor subunit changes is of great potential use...... for studies of excitotoxic, glutamate receptor-induced neuronal cell death, receptor modulation and related neuroprotection....

  7. The BRAIN Initiative Cell Census Consortium: Lessons Learned toward Generating a Comprehensive Brain Cell Atlas.

    Science.gov (United States)

    Ecker, Joseph R; Geschwind, Daniel H; Kriegstein, Arnold R; Ngai, John; Osten, Pavel; Polioudakis, Damon; Regev, Aviv; Sestan, Nenad; Wickersham, Ian R; Zeng, Hongkui

    2017-11-01

    A comprehensive characterization of neuronal cell types, their distributions, and patterns of connectivity is critical for understanding the properties of neural circuits and how they generate behaviors. Here we review the experiences of the BRAIN Initiative Cell Census Consortium, ten pilot projects funded by the U.S. BRAIN Initiative, in developing, validating, and scaling up emerging genomic and anatomical mapping technologies for creating a complete inventory of neuronal cell types and their connections in multiple species and during development. These projects lay the foundation for a larger and longer-term effort to generate whole-brain cell atlases in species including mice and humans. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. Henrietta Lacks, HeLa cells, and cell culture contamination.

    Science.gov (United States)

    Lucey, Brendan P; Nelson-Rees, Walter A; Hutchins, Grover M

    2009-09-01

    Henrietta Lacks died in 1951 of an aggressive adenocarcinoma of the cervix. A tissue biopsy obtained for diagnostic evaluation yielded additional tissue for Dr George O. Gey's tissue culture laboratory at Johns Hopkins (Baltimore, Maryland). The cancer cells, now called HeLa cells, grew rapidly in cell culture and became the first human cell line. HeLa cells were used by researchers around the world. However, 20 years after Henrietta Lacks' death, mounting evidence suggested that HeLa cells contaminated and overgrew other cell lines. Cultures, supposedly of tissues such as breast cancer or mouse, proved to be HeLa cells. We describe the history behind the development of HeLa cells, including the first published description of Ms Lacks' autopsy, and the cell culture contamination that resulted. The debate over cell culture contamination began in the 1970s and was not harmonious. Ultimately, the problem was not resolved and it continues today. Finally, we discuss the philosophical implications of the immortal HeLa cell line.

  9. Flux analysis of mammalian cell culture

    NARCIS (Netherlands)

    Martens, D.E.; Tramper, J.

    2010-01-01

    Animal cells are used for the production of vaccines and pharmaceutical proteins. The increase in demand for these products requires an increase in volumetric productivity of animal cell culture processes, which can be attained through an increase in biomass concentration and/or specific

  10. Human cell culture in a space bioreactor

    Science.gov (United States)

    Morrison, Dennis R.

    1988-01-01

    Microgravity offers new ways of handling fluids, gases, and growing mammalian cells in efficient suspension cultures. In 1976 bioreactor engineers designed a system using a cylindrical reactor vessel in which the cells and medium are slowly mixed. The reaction chamber is interchangeable and can be used for several types of cell cultures. NASA has methodically developed unique suspension type cell and recovery apparatus culture systems for bioprocess technology experiments and production of biological products in microgravity. The first Space Bioreactor was designed for microprocessor control, no gaseous headspace, circulation and resupply of culture medium, and slow mixing in very low shear regimes. Various ground based bioreactors are being used to test reactor vessel design, on-line sensors, effects of shear, nutrient supply, and waste removal from continuous culture of human cells attached to microcarriers. The small Bioreactor is being constructed for flight experiments in the Shuttle Middeck to verify systems operation under microgravity conditions and to measure the efficiencies of mass transport, gas transfer, oxygen consumption and control of low shear stress on cells.

  11. The presence of serum alters the properties of iron oxide nanoparticles and lowers their accumulation by cultured brain astrocytes

    Science.gov (United States)

    Geppert, Mark; Petters, Charlotte; Thiel, Karsten; Dringen, Ralf

    2013-01-01

    Iron oxide nanoparticles (IONPs) are considered for various diagnostic and therapeutic applications. Such particles are able to cross the blood-brain barrier and are taken up into brain cells. To test whether serum components affect the properties of IONPs and/or their uptake into brain cells, we have incubated dimercaptosuccinate-coated magnetic IONPs without and with fetal calf serum (FCS) and have exposed cultured brain astrocytes with IONPs in the absence or presence of FCS. Incubation with FCS caused a concentration-dependent increase in the average hydrodynamic diameter of the particles and of their zeta-potential. In the presence of 10 % FCS, the diameter of the IONPs increased from 57 ± 2 to 107 ± 6 nm and the zeta-potential of the particles from -22 ± 5 to -9 ± 1 mV. FCS affected also strongly the uptake of IONPs by cultured astrocytes. The efficient time- and temperature-dependent cellular accumulation of IONPs was lowered with increasing concentration of FCS by up to 90 %. In addition, in the absence of serum, endocytosis inhibitors did not alter the IONP accumulation by astrocytes, while chlorpromazine or wortmannin lowered significantly the accumulation of IONPs in the presence of FCS, suggesting that clathrin-mediated endocytosis and macropinocytosis are involved in astrocytic IONP uptake from serum-containing medium. These data demonstrate that the presence of FCS strongly affects the properties of IONPs as well as their accumulation by cultured brain cells.

  12. The presence of serum alters the properties of iron oxide nanoparticles and lowers their accumulation by cultured brain astrocytes

    Energy Technology Data Exchange (ETDEWEB)

    Geppert, Mark; Petters, Charlotte [University of Bremen, Centre for Biomolecular Interactions Bremen (Germany); Thiel, Karsten [Fraunhofer Institute for Manufacturing Technology and Advanced Materials (Germany); Dringen, Ralf, E-mail: ralf.dringen@uni-bremen.de [University of Bremen, Centre for Biomolecular Interactions Bremen (Germany)

    2013-01-15

    Iron oxide nanoparticles (IONPs) are considered for various diagnostic and therapeutic applications. Such particles are able to cross the blood-brain barrier and are taken up into brain cells. To test whether serum components affect the properties of IONPs and/or their uptake into brain cells, we have incubated dimercaptosuccinate-coated magnetic IONPs without and with fetal calf serum (FCS) and have exposed cultured brain astrocytes with IONPs in the absence or presence of FCS. Incubation with FCS caused a concentration-dependent increase in the average hydrodynamic diameter of the particles and of their zeta-potential. In the presence of 10 % FCS, the diameter of the IONPs increased from 57 {+-} 2 to 107 {+-} 6 nm and the zeta-potential of the particles from -22 {+-} 5 to -9 {+-} 1 mV. FCS affected also strongly the uptake of IONPs by cultured astrocytes. The efficient time- and temperature-dependent cellular accumulation of IONPs was lowered with increasing concentration of FCS by up to 90 %. In addition, in the absence of serum, endocytosis inhibitors did not alter the IONP accumulation by astrocytes, while chlorpromazine or wortmannin lowered significantly the accumulation of IONPs in the presence of FCS, suggesting that clathrin-mediated endocytosis and macropinocytosis are involved in astrocytic IONP uptake from serum-containing medium. These data demonstrate that the presence of FCS strongly affects the properties of IONPs as well as their accumulation by cultured brain cells.

  13. Organotypic brain slice cultures of adult transgenic P301S mice--a model for tauopathy studies.

    Directory of Open Access Journals (Sweden)

    Agneta Mewes

    Full Text Available BACKGROUND: Organotypic brain slice cultures represent an excellent compromise between single cell cultures and complete animal studies, in this way replacing and reducing the number of animal experiments. Organotypic brain slices are widely applied to model neuronal development and regeneration as well as neuronal pathology concerning stroke, epilepsy and Alzheimer's disease (AD. AD is characterized by two protein alterations, namely tau hyperphosphorylation and excessive amyloid β deposition, both causing microglia and astrocyte activation. Deposits of hyperphosphorylated tau, called neurofibrillary tangles (NFTs, surrounded by activated glia are modeled in transgenic mice, e.g. the tauopathy model P301S. METHODOLOGY/PRINCIPAL FINDINGS: In this study we explore the benefits and limitations of organotypic brain slice cultures made of mature adult transgenic mice as a potential model system for the multifactorial phenotype of AD. First, neonatal (P1 and adult organotypic brain slice cultures from 7- to 10-month-old transgenic P301S mice have been compared with regard to vitality, which was monitored with the lactate dehydrogenase (LDH- and the MTT (3-(4,5-Dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide assays over 15 days. Neonatal slices displayed a constant high vitality level, while the vitality of adult slice cultures decreased significantly upon cultivation. Various preparation and cultivation conditions were tested to augment the vitality of adult slices and improvements were achieved with a reduced slice thickness, a mild hypothermic cultivation temperature and a cultivation CO(2 concentration of 5%. Furthermore, we present a substantial immunohistochemical characterization analyzing the morphology of neurons, astrocytes and microglia in comparison to neonatal tissue. CONCLUSION/SIGNIFICANCE: Until now only adolescent animals with a maximum age of two months have been used to prepare organotypic brain slices. The current study

  14. Increasing cell culture population doublings for long-term growth of finite life span human cell cultures

    Science.gov (United States)

    Stampfer, Martha R; Garbe, James C

    2015-02-24

    Cell culture media formulations for culturing human epithelial cells are herein described. Also described are methods of increasing population doublings in a cell culture of finite life span human epithelial cells and prolonging the life span of human cell cultures. Using the cell culture media disclosed alone and in combination with addition to the cell culture of a compound associated with anti-stress activity achieves extended growth of pre-stasis cells and increased population doublings and life span in human epithelial cell cultures.

  15. Biosynthesis of the D2 cell adhesion molecule: pulse-chase studies in cultured fetal rat neuronal cells

    DEFF Research Database (Denmark)

    Lyles, J M; Norrild, B; Bock, E

    1984-01-01

    D2 is a membrane glycoprotein that is believed to function as a cell adhesion molecule (CAM) in neural cells. We have examined its biosynthesis in cultured fetal rat brain neurones. We found D2-CAM to be synthesized initially as two polypeptides: Mr 186,000 (A) and Mr 136,000 (B). With increasing...

  16. Oscar Wilde and the brain cell.

    Science.gov (United States)

    Cohn, Elisha

    2013-01-01

    This chapter considers Oscar Wilde's interest in the brain cell as an aesthetic object. Offering an account of Wilde's career that analyzes his early interest in physiology and philosophy, this chapter argues that Wilde's uniquely aesthetic take on the brain suggests that he rejects an account of the self as autonomous or self-determining. For many late Victorians brain science threatened both the freedom of human action and the legitimacy of beauty because it had the potential to invalidate conscious experience. But writers whose work Wilde knew, like John Ruskin, W. K. Clifford, and John Tyndall, avoided the despair of materialism by using aesthetic terms in their own discussions of life's invisible materials. Wilde's art collaborates with the contemporary sciences. His depictions of the cell direct the senses to a new field of being that emphasizes the molecular life all humans have in common, in which individual responsibility and activity matter less than the necessity of beauty. © 2013 Elsevier B.V. All rights reserved.

  17. Perturbation of myelination by activation of distinct signaling pathways : An in vitro study in a myelinating culture derived from fetal rat brain

    NARCIS (Netherlands)

    Baron, W; de Jonge, JC; Vries, de Hans; Hoekstra, D

    2000-01-01

    An in vitro myelinating mouse-derived model system has been adapted and optimized for fetal rat brain. In these mixed brain cell (MBC) cultures, myelinogenesis was studied by examining the effect of signaling pathways that are involved in the timing of oligodendrocyte differentiation. When PMA, a

  18. Isolation of a Pluripotent Neural Stem Cell from the Embryonic Bovine Brain

    Directory of Open Access Journals (Sweden)

    Yuhua Gao

    2015-03-01

    Full Text Available We recently isolated stem cells derived from the brain of a bovine fetus, utilizing a particular mechanical separation method. After improving our experimental conditions, we obtained neural stem cells using an optimized culture medium system. The cells were expanded, established in continuous cell culture and used for immunofluorescence cytochemistry. RT-PCR showed that embryonic neural stem cells (NSCs not only expresses the protein Sox2, Nestin but also Pax6, Musashi proteins and were differentiated into the three classical neuronal phenotypes (neurons, astrocytes, and oligodendrocytes.

  19. 21 CFR 864.2280 - Cultured animal and human cells.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Cultured animal and human cells. 864.2280 Section... (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Cell And Tissue Culture Products § 864.2280 Cultured animal and human cells. (a) Identification. Cultured animal and human cells are in vitro...

  20. Cell Culture on MEMS Platforms: A Review

    Science.gov (United States)

    Ni, Ming; Tong, Wen Hao; Choudhury, Deepak; Rahim, Nur Aida Abdul; Iliescu, Ciprian; Yu, Hanry

    2009-01-01

    Microfabricated systems provide an excellent platform for the culture of cells, and are an extremely useful tool for the investigation of cellular responses to various stimuli. Advantages offered over traditional methods include cost-effectiveness, controllability, low volume, high resolution, and sensitivity. Both biocompatible and bio-incompatible materials have been developed for use in these applications. Biocompatible materials such as PMMA or PLGA can be used directly for cell culture. However, for bio-incompatible materials such as silicon or PDMS, additional steps need to be taken to render these materials more suitable for cell adhesion and maintenance. This review describes multiple surface modification strategies to improve the biocompatibility of MEMS materials. Basic concepts of cell-biomaterial interactions, such as protein adsorption and cell adhesion are covered. Finally, the applications of these MEMS materials in Tissue Engineering are presented. PMID:20054478

  1. Vitamin B-complex initiates growth and development of human embryonic brain cells in vitro.

    Science.gov (United States)

    Danielyan, K E; Abramyan, R A; Galoyan, A A; Kevorkian, G A

    2011-09-01

    We studied a combined effect of subcomponents of vitamin B complex on the growth, development, and death of human embryonic brain-derived cells (E90) cultured using a modified method of Matson. Cell death was detected by trypan blue staining. According to our results, vitamin B-complex in low-doses promote the development, maturation, and enlargement of human embryonic brain cells, on the one hand, and increases the percent of cell death, which attests to accelerated maturation and metabolism, on the other.

  2. Proliferation of cultured mouse choroid plexus epithelial cells.

    Directory of Open Access Journals (Sweden)

    Basam Z Barkho

    Full Text Available The choroid plexus (ChP epithelium is a multifunctional tissue found in the ventricles of the brain. The major function of the ChP epithelium is to produce cerebrospinal fluid (CSF that bathes and nourishes the central nervous system (CNS. In addition to the CSF, ChP epithelial cells (CPECs produce and secrete numerous neurotrophic factors that support brain homeostasis, such as adult hippocampal neurogenesis. Accordingly, damage and dysfunction to CPECs are thought to accelerate and intensify multiple disease phenotypes, and CPEC regeneration would represent a potential therapeutic approach for these diseases. However, previous reports suggest that CPECs rarely divide, although this has not been extensively studied in response to extrinsic factors. Utilizing a cell-cycle reporter mouse line and live cell imaging, we identified scratch injury and the growth factors insulin-like growth factor 1 (IGF-1 and epidermal growth factor (EGF as extrinsic cues that promote increased CPEC expansion in vitro. Furthermore, we found that IGF-1 and EGF treatment enhances scratch injury-induced proliferation. Finally, we established whole tissue explant cultures and observed that IGF-1 and EGF promote CPEC division within the intact ChP epithelium. We conclude that although CPECs normally have a slow turnover rate, they expand in response to external stimuli such as injury and/or growth factors, which provides a potential avenue for enhancing ChP function after brain injury or neurodegeneration.

  3. Effects of radiation on cultured fish cells

    International Nuclear Information System (INIS)

    Etoh, Hisami; Suyama, Ippei

    1980-01-01

    A new fibroblastic cell line was established in our laboratory from the caudal fin of the goldfish, C. auratus. The cells, designated CAF, have been subcultured over 80 passages since initiation in August, 1977. A brief description of cell cultivation and colony formation is presented. The plating efficiency obtained was considerably higher than those reported for other fish cell lines. CAF cells were irradiated with 250, 500, 1,000, 2,000, and 3,000 R of x-rays at a dose rate of 80 R/min in air. The survival parameters changed when the number of passages of culture increased. Values for D 0 , D sub(q), and n obtained from cells irradiated at the 70th passage were calculated to be 650 R, 700 R, and 2.7 respectively. Thus CAF cells would be several times as resistant in general as cultured mammalian cells. The cells irradiated with 1,000 R of x-rays received a second dose from 250 to 2,000 R at intervals of 3, 6, and 24 hr. The cells kept at 26 0 C showed a pronounced recovery from sublethal damage during the intervals between two doses. Magnitude of recovery was larger if the interval was longer under the present experimental conditions. These results may indicate that the recovery observed at an individual level accounts partly for that in vitro. (author)

  4. The metabolism of malate by cultured rat brain astrocytes

    Energy Technology Data Exchange (ETDEWEB)

    McKenna, M.C.; Tildon, J.T.; Couto, R.; Stevenson, J.H.; Caprio, F.J. (Department of Pediatrics, University of Maryland School of Medicine, Baltimore (USA))

    1990-12-01

    Since malate is known to play an important role in a variety of functions in the brain including energy metabolism, the transfer of reducing equivalents and possibly metabolic trafficking between different cell types; a series of biochemical determinations were initiated to evaluate the rate of 14CO2 production from L-(U-14C)malate in rat brain astrocytes. The 14CO2 production from labeled malate was almost totally suppressed by the metabolic inhibitors rotenone and antimycin A suggesting that most of malate metabolism was coupled to the electron transport system. A double reciprocal plot of the 14CO2 production from the metabolism of labeled malate revealed biphasic kinetics with two apparent Km and Vmax values suggesting the presence of more than one mechanism of malate metabolism in these cells. Subsequent experiments were carried out using 0.01 mM and 0.5 mM malate to determine whether the addition of effectors would differentially alter the metabolism of high and low concentrations of malate. Effectors studied included compounds which could be endogenous regulators of malate metabolism and metabolic inhibitors which would provide information regarding the mechanisms regulating malate metabolism. Both lactate and aspartate decreased 14CO2 production from malate equally. However, a number of effectors were identified which selectively altered the metabolism of 0.01 mM malate including aminooxyacetate, furosemide, N-acetylaspartate, oxaloacetate, pyruvate and glucose, but had little or no effect on the metabolism of 0.5 mM malate. In addition, alpha-ketoglutarate and succinate decreased 14CO2 production from 0.01 mM malate much more than from 0.5 mM malate. In contrast, a number of effectors altered the metabolism of 0.5 mM malate more than 0.01 mM. These included methionine sulfoximine, glutamate, malonate, alpha-cyano-4-hydroxycinnamate and ouabain.

  5. Characterization of Cancer Stem Cells in Patients with Brain ...

    African Journals Online (AJOL)

    Background: Gliomas, in general, and astrocytomas, in particular, represent the most frequent primary brain tumors. Nowadays, it is increasingly believed that gliomas may arise from cancer stem cells, which share several characteristics with normal neural stem cells. Brain tumor stem cells have been found to express a ...

  6. Embryo forming cells in carrot suspension cultures

    NARCIS (Netherlands)

    Toonen, M.A.J.

    1997-01-01


    Somatic cells of many plant species can be cultured in vitro and induced to form embryos that are able to develop into mature plants. This process, termed somatic embryogenesis, was originally described in carrot (Daucus carota L.). Somatic embryos develop through the same characteristic

  7. Nanotechnology, Cell Culture and Tissue Engineering

    Directory of Open Access Journals (Sweden)

    Kazutoshi Haraguchi

    2011-01-01

    Full Text Available We have fabricated new types of polymer hydrogels and polymer nanocomposites, i.e., nanocomposite gels (NC gels and soft, polymer nanocomposites (M-NCs: solid, with novel organic/inorganic network structures. Both NC gels and M-NCs were synthesized by in-situ free-radical polymerization in the presence of exfoliated clay platelets in aqueous systems and were obtained in various forms such as film, sheet, tube, coating, etc. and sizes with a wide range of clay contents. Here, disk-like inorganic clay nanoparticles act as multi-functional crosslinkers to form new types of network systems. Both NC gels and M-NCs have extraordinary optical and mechanical properties including ultra-high reversible extensibility, as well as a number of new characteristics relating to optical anisotropy, polymer/clay morphology, biocompatibility, stimuli-sensitive surfaces, micro-patterning, etc. For examples, the biological testing of medical devices, comprised of a sensitization test, an irritation test, an intracutaneous test and an in vitro cytotoxicity test,was carried out for NC gels and M-NCs. The safety of NC gels and M-NCs was confirmed in all tests. Also, the interaction of living tissue with NC gel was investigated in vivo by implantation in live goats; neither inflammation nor concrescence occurred around the NC gels. Furthermore, it was found that both N-NC gels consisting of poly(N-isopropylacrylamide(PNIPA/clay network and M-NCs consisting of poly(2-methoxyethyacrylate(PMEA/clay network show characteristic cell culture and subsequent cell detachment on their surfaces, although it was almost impossible to culture cells on conventional, chemically-crosslinked PNIPA hydrogels and chemically crossslinked PMEA, regardless of their crosslinker concentration. Various kinds of cells, such ashumanhepatoma cells (HepG2, normal human dermal fibroblast (NHDF, and human umbilical vein endothelial cells (HUVEC, could be cultured to be confluent on the surfaces of N

  8. Bioactive form of resveratrol in glioblastoma cells and its safety for normal brain cells

    Directory of Open Access Journals (Sweden)

    Xiao-Hong Shu

    2013-05-01

    Full Text Available ABSTRACTBackground: Resveratrol, a plant polyphenol existing in grapes and many other natural foods, possesses a wide range of biological activities including cancer prevention. It has been recognized that resveratrol is intracellularly biotransformed to different metabolites, but no direct evidence has been available to ascertain its bioactive form because of the difficulty to maintain resveratrol unmetabolized in vivo or in vitro. It would be therefore worthwhile to elucidate the potential therapeutic implications of resveratrol metabolism using a reliable resveratrol-sensitive cancer cells.Objective: To identify the real biological form of trans-resveratrol and to evaluate the safety of the effective anticancer dose of resveratrol for the normal brain cells.Methods: The samples were prepared from the condition media and cell lysates of human glioblastoma U251 cells, and were purified by solid phase extraction (SPE. The samples were subjected to high performance liquid chromatography (HPLC and liquid chromatography/tandem mass spectrometry (LC/MS analysis. According to the metabolite(s, trans-resveratrol was biotransformed in vitro by the method described elsewhere, and the resulting solution was used to treat U251 cells. Meanwhile, the responses of U251 and primarily cultured rat normal brain cells (glial cells and neurons to 100μM trans-resveratrol were evaluated by multiple experimental methods.Results: The results revealed that resveratrol monosulfate was the major metabolite in U251 cells. About half fraction of resveratrol monosulfate was prepared in vitro and this trans-resveratrol and resveratrol monosulfate mixture showed little inhibitory effect on U251 cells. It is also found that rat primary brain cells (PBCs not only resist 100μM but also tolerate as high as 200μM resveratrol treatment.Conclusions: Our study thus demonstrated that trans-resveratrol was the bioactive form in glioblastoma cells and, therefore, the biotransforming

  9. Mitochondrial activity assessed by cytofluorescence after in-vitro-irradiation of primary rat brain cultures

    International Nuclear Information System (INIS)

    Cervos-Navarro, J.; Hamdorf, G.

    1993-01-01

    Mitochondria play a key role in cell homeostasis and are the first cell organells affected by ionizing irradiation, as it was proved by previous electron microscopic investigations. In order to observe functional parameters of mitochondria after low-dose irradiation, primary rat brain cultures (prepared from 15-day-old rat fetuses) were irradiated from a 60 Co-source with 0.5 and 1 Gy at the age of 2 or 7 days in vitro (div). Cytofluorescence measurement was made by a Cytofluor trademark2350 using Rhodamine 123. This fluorescent dye is positively charged and accumulates specifically in the mitochondria of living cells without cytotoxic effect. Since its retention depends on the negative membrane potential as well as the proton gradient that exists across the inner mitochondrial membrane, Rhodamine 123 accumulation reflects the status of mitochondrial activity as a whole. After irradiation with 0.5 and 1 Gy on day 2 in culture there was a decrease in Rhodamine uptake in the irradiated cultures during the first week after the irradiation insult which reached minimum values after 3 days. Rhodamine uptake increased during the following period and finally reached the values of the control cultures. In the second experiment with irradiated cultures on day 7 and the same doses of 0.5 and 1 Gy the accumulation of Rhodamine decreased only initially then increased tremendously. After both doses values of Rhodamine-accumulation were higher than the control level. The results demonstrated that irradiation caused a change in mitochondrial activity depending on the time of irradiation. The dramatic increase over the control levels after irradiation on day 7 in vitro is attributed to the fact that at this time synapses have already developed. Deficiency of mitochondrial activity as well as hyperactivity and the consequent change in energy production may lead to changes in neuronal metabolism including an increase in production of free radicals

  10. Cell Culture Assay for Human Noroviruses [response

    Energy Technology Data Exchange (ETDEWEB)

    Straub, Tim M.; Honer Zu Bentrup, Kerstin; Orosz Coghlan, Patricia; Dohnalkova, Alice; Mayer, Brooke K.; Bartholomew, Rachel A.; Valdez, Catherine O.; Bruckner-Lea, Cindy J.; Gerba, Charles P.; Abbaszadegan, Morteza A.; Nickerson, Cheryl A.

    2007-07-01

    We appreciate the comments provided by Leung et al., in response to our recently published article “In Vitro Cell Culture Infectivity Assay for Human Noroviruses” by Straub et al. (1). The specific aim of our project was to develop an in vitro cell culture infectivity assay for human noroviruses (hNoV) to enhance risk assessments when they are detected in water supplies. Reverse transcription (RT) qualitative or quantitative PCR are the primary assays for waterborne NoV monitoring. However, these assays cannot distinguish between infectious vs. non-infectious virions. When hNoV is detected in water supplies, information provided by our infectivity assay will significantly improve risk assessment models and protect human health, regardless of whether we are propagating NoV. Indeed, in vitro cell culture infectivity assays for the waterborne pathogen Cryptosporidium parvum that supplement approved fluorescent microscopy assays, do not result in amplification of the environmentally resistant hard-walled oocysts (2). However, identification of life cycle stages in cell culture provides evidence of infectious oocysts in a water supply. Nonetheless, Leung et al.’s assertion regarding the suitability of our method for the in vitro propagation of high titers of NoV is valid for the medical research community. In this case, well-characterized challenge pools of virus would be useful for developing and testing diagnostics, therapeutics, and vaccines. As further validation of our published findings, we have now optimized RT quantitative PCR to assess the level of viral production in cell culture, where we are indeed finding significant increases in viral titer. The magnitude and time course of these increases is dependent on both virus strain and multiplicity of infection. We are currently preparing a manuscript that will discuss these findings in greater detail, and the implications this may have for creating viral challenge pools

  11. Radiation response of cultured human cells is unaffected by Johrei.

    Science.gov (United States)

    Hall, Zach; Luu, Tri; Moore, Dan; Yount, Garret

    2007-06-01

    Johrei has been credited with healing thousands from radiation wounds after the Hiroshima and Nagasaki bombs in 1945. This alternative medical therapy is becoming increasingly popular in the United States, as are other Energy Medicine modalities that purport to influence a universal healing energy. Human brain cells were cultured and exposed to increasing doses of ionizing radiation. Experienced Johrei practitioners directed healing intentionality toward the cells for 30 min from a distance of 20 cm and the fate of the cells was observed by computerized time-lapse microscopy. Cell death and cell divisions were tallied every 30 min before, during and after Johrei treatment for a total of 22.5 h. An equal number of control experiments were conducted in which cells were irradiated but did not receive Johrei treatment. Samples were assigned to treatment conditions randomly and data analysis was conducted in a blinded fashion. Radiation exposure decreased the rate of cell division (cell cycle arrest) in a dose-dependent manner. Division rates were estimated for each 30 min and averaged over 8 independent experiments (4 control and 4 with Johrei treatment) for each of 4 doses of X-rays (0, 2, 4 and 8 Gy). Because few cell deaths were observed, pooled data from the entire observation period were used to estimate death rates. Analysis of variance did not reveal any significant differences on division rate or death rate between treatment groups. Only radiation dose was statistically significant. We found no indication that the radiation response of cultured cells is affected by Johrei treatment.

  12. Radiation Response of Cultured Human Cells Is Unaffected by Johrei

    Directory of Open Access Journals (Sweden)

    Zach Hall

    2007-01-01

    Full Text Available Johrei has been credited with healing thousands from radiation wounds after the Hiroshima and Nagasaki bombs in 1945. This alternative medical therapy is becoming increasingly popular in the United States, as are other Energy Medicine modalities that purport to influence a universal healing energy. Human brain cells were cultured and exposed to increasing doses of ionizing radiation. Experienced Johrei practitioners directed healing intentionality toward the cells for 30 min from a distance of 20 cm and the fate of the cells was observed by computerized time-lapse microscopy. Cell death and cell divisions were tallied every 30 min before, during and after Johrei treatment for a total of 22.5 h. An equal number of control experiments were conducted in which cells were irradiated but did not receive Johrei treatment. Samples were assigned to treatment conditions randomly and data analysis was conducted in a blinded fashion. Radiation exposure decreased the rate of cell division (cell cycle arrest in a dose-dependent manner. Division rates were estimated for each 30 min and averaged over 8 independent experiments (4 control and 4 with Johrei treatment for each of 4 doses of X-rays (0, 2, 4 and 8 Gy. Because few cell deaths were observed, pooled data from the entire observation period were used to estimate death rates. Analysis of variance did not reveal any significant differences on division rate or death rate between treatment groups. Only radiation dose was statistically significant. We found no indication that the radiation response of cultured cells is affected by Johrei treatment.

  13. Electrical Guidance of Human Stem Cells in the Rat Brain

    Directory of Open Access Journals (Sweden)

    Jun-Feng Feng

    2017-07-01

    Full Text Available Limited migration of neural stem cells in adult brain is a roadblock for the use of stem cell therapies to treat brain diseases and injuries. Here, we report a strategy that mobilizes and guides migration of stem cells in the brain in vivo. We developed a safe stimulation paradigm to deliver directional currents in the brain. Tracking cells expressing GFP demonstrated electrical mobilization and guidance of migration of human neural stem cells, even against co-existing intrinsic cues in the rostral migration stream. Transplanted cells were observed at 3 weeks and 4 months after stimulation in areas guided by the stimulation currents, and with indications of differentiation. Electrical stimulation thus may provide a potential approach to facilitate brain stem cell therapies.

  14. Taurine Induces Proliferation of Neural Stem Cells and Synapse Development in the Developing Mouse Brain

    Science.gov (United States)

    Shivaraj, Mattu Chetana; Marcy, Guillaume; Low, Guoliang; Ryu, Jae Ryun; Zhao, Xianfeng; Rosales, Francisco J.; Goh, Eyleen L. K.

    2012-01-01

    Taurine is a sulfur-containing amino acid present in high concentrations in mammalian tissues. It has been implicated in several processes involving brain development and neurotransmission. However, the role of taurine in hippocampal neurogenesis during brain development is still unknown. Here we show that taurine regulates neural progenitor cell (NPC) proliferation in the dentate gyrus of the developing brain as well as in cultured early postnatal (P5) hippocampal progenitor cells and hippocampal slices derived from P5 mice brains. Taurine increased cell proliferation without having a significant effect on neural differentiation both in cultured P5 NPCs as well as cultured hippocampal slices and in vivo. Expression level analysis of synaptic proteins revealed that taurine increases the expression of Synapsin 1 and PSD 95. We also found that taurine stimulates the phosphorylation of ERK1/2 indicating a possible role of the ERK pathway in mediating the changes that we observed, especially in proliferation. Taken together, our results demonstrate a role for taurine in neural stem/progenitor cell proliferation in developing brain and suggest the involvement of the ERK1/2 pathways in mediating these actions. Our study also shows that taurine influences the levels of proteins associated with synapse development. This is the first evidence showing the effect of taurine on early postnatal neuronal development using a combination of in vitro, ex-vivo and in vivo systems. PMID:22916184

  15. Taurine induces proliferation of neural stem cells and synapse development in the developing mouse brain.

    Directory of Open Access Journals (Sweden)

    Mattu Chetana Shivaraj

    Full Text Available Taurine is a sulfur-containing amino acid present in high concentrations in mammalian tissues. It has been implicated in several processes involving brain development and neurotransmission. However, the role of taurine in hippocampal neurogenesis during brain development is still unknown. Here we show that taurine regulates neural progenitor cell (NPC proliferation in the dentate gyrus of the developing brain as well as in cultured early postnatal (P5 hippocampal progenitor cells and hippocampal slices derived from P5 mice brains. Taurine increased cell proliferation without having a significant effect on neural differentiation both in cultured P5 NPCs as well as cultured hippocampal slices and in vivo. Expression level analysis of synaptic proteins revealed that taurine increases the expression of Synapsin 1 and PSD 95. We also found that taurine stimulates the phosphorylation of ERK1/2 indicating a possible role of the ERK pathway in mediating the changes that we observed, especially in proliferation. Taken together, our results demonstrate a role for taurine in neural stem/progenitor cell proliferation in developing brain and suggest the involvement of the ERK1/2 pathways in mediating these actions. Our study also shows that taurine influences the levels of proteins associated with synapse development. This is the first evidence showing the effect of taurine on early postnatal neuronal development using a combination of in vitro, ex-vivo and in vivo systems.

  16. Use of an adaptable cell culture kit for performing lymphocyte and monocyte cell cultures in microgravity

    Science.gov (United States)

    Hatton, J. P.; Lewis, M. L.; Roquefeuil, S. B.; Chaput, D.; Cazenave, J. P.; Schmitt, D. A.

    1998-01-01

    The results of experiments performed in recent years on board facilities such as the Space Shuttle/Spacelab have demonstrated that many cell systems, ranging from simple bacteria to mammalian cells, are sensitive to the microgravity environment, suggesting gravity affects fundamental cellular processes. However, performing well-controlled experiments aboard spacecraft offers unique challenges to the cell biologist. Although systems such as the European 'Biorack' provide generic experiment facilities including an incubator, on-board 1-g reference centrifuge, and contained area for manipulations, the experimenter must still establish a system for performing cell culture experiments that is compatible with the constraints of spaceflight. Two different cell culture kits developed by the French Space Agency, CNES, were recently used to perform a series of experiments during four flights of the 'Biorack' facility aboard the Space Shuttle. The first unit, Generic Cell Activation Kit 1 (GCAK-1), contains six separate culture units per cassette, each consisting of a culture chamber, activator chamber, filtration system (permitting separation of cells from supernatant in-flight), injection port, and supernatant collection chamber. The second unit (GCAK-2) also contains six separate culture units, including a culture, activator, and fixation chambers. Both hardware units permit relatively complex cell culture manipulations without extensive use of spacecraft resources (crew time, volume, mass, power), or the need for excessive safety measures. Possible operations include stimulation of cultures with activators, separation of cells from supernatant, fixation/lysis, manipulation of radiolabelled reagents, and medium exchange. Investigations performed aboard the Space Shuttle in six different experiments used Jurkat, purified T-cells or U937 cells, the results of which are reported separately. We report here the behaviour of Jurkat and U937 cells in the GCAK hardware in ground

  17. Lipoprotein receptors in cultured bovine endothelial cells

    International Nuclear Information System (INIS)

    Struempfer, A.E.M.

    1983-07-01

    In this study, receptors that may be involved in the uptake of low density lipoproteins (LDL) and low density lipoproteins which have been modified by acetylation (AcLDL), were characterized. Aortic epithelial cells were used and a cell culture system which closely resembled the in vivo monolayer was established. Endothelial cell and lipoprotein interactions were examined by incubating the cells with 125 l-labelled lipoproteins under various conditions. The receptor affinity of bovine aortic endothelial cells was higher for AcLDL than that for LDL. Competition studies demonstrated that there were two distinct receptors for LDL and AcLDL on the endothelial cells. AcLDL did not compete with LDL for the LDL receptor, and conversely LDL did not compete with AcLDL for the AcLDL receptor. The receptor activities for LDL and AcLDL were examined as a function of culture age. Whereas the LDL receptor could be regulated, the AcLDL receptor was not as susceptible to regulation. Upon exposing endothelial cells for 72 h to either LDL or AcLDL, it was found that the total amount of cellular cholesterol increased by about 50%. However, the increase of total cholesterol was largely in the form of free cholesterol. This is in contrast to macrophages, where the increase in total cholesterol upon exposure to AcLDL is largely in the form cholesteryl esters

  18. Dynamic cell culture system (7-IML-1)

    Science.gov (United States)

    Cogoli, Augusto

    1992-01-01

    This experiment is one of the Biorack experiments being flown on the International Microgravity Laboratory 1 (MIL-1) mission as part of an investigation studying cell proliferation and performance in space. One of the objectives of this investigation is to assess the potential benefits of bioprocessing in space with the ultimate goal of developing a bioreactor for continuous cell cultures in space. This experiment will test the operation of an automated culture chamber that was designed for use in a Bioreactor in space. The device to be tested is called the Dynamic Cell Culture System (DCCS). It is a simple device in which media are renewed or chemicals are injected automatically, by means of osmotic pumps. This experiment uses four Type I/O experiment containers. One DCCS unit, which contains a culture chamber with renewal of medium and a second chamber without a medium supply fits in each container. Two DCCS units are maintained under zero gravity conditions during the on-orbit period. The other two units are maintained under 1 gh conditions in a 1 g centrifuge. The schedule for incubator transfer is given.

  19. A biocompatible micro cell culture chamber (mu CCC) for the culturing and on-line monitoring of eukaryote cells

    DEFF Research Database (Denmark)

    Stangegaard, Michael; Petronis, Sarunas; Jørgensen, Anders Michael

    2006-01-01

    on cell survival. Low grade light exposure was however compatible with optical recordings as well as cell viability. These results strongly indicate that a cell culture chip could be constructed that allowed for on-line optical recording of cellular events without affecting the cell culturing condition...... culture chip compared to cell culture flasks. The cell culture chip could without further modification support cell growth of two other cell lines. Light coming from the microscope lamp during optical recordings of the cells was the only external factor identified, that could have a negative effect...

  20. Imaging of human glioblastoma cells and their interactions with mesenchymal stem cells in the zebrafish (Danio rerio) embryonic brain

    International Nuclear Information System (INIS)

    Vittori, Milos; Breznik, Barbara; Gredar, Tajda; Hrovat, Katja; Bizjak Mali, Lilijana; Lah, Tamara T

    2016-01-01

    An attractive approach in the study of human cancers is the use of transparent zebrafish (Danio rerio) embryos, which enable the visualization of cancer progression in a living animal. We implanted mixtures of fluorescently labeled glioblastoma (GBM) cells and bonemarrow-derived mesenchymal stem cells (MSCs) into zebrafish embryos to study the cellular pathways of their invasion and the interactions between these cells in vivo. By developing and applying a carbocyanine-dye-compatible clearing protocol for observation of cells in deep tissues, we showed that U87 and U373 GBM cells rapidly aggregated into tumor masses in the ventricles and midbrain hemispheres of the zebrafish embryo brain, and invaded the central nervous system, often using the ventricular system and the central canal of the spinal cord. However, the GBM cells did not leave the central nervous system. With co-injection of differentially labeled cultured GBM cells and MSCs, the implanted cells formed mixed tumor masses in the brain. We observed tight associations between GBM cells and MSCs, and possible cell-fusion events. GBM cells and MSCs used similar invasion routes in the central nervous system. This simple model can be used to study the molecular pathways of cellular processes in GBM cell invasion, and their interactions with various types of stromal cells in double or triple cell co-cultures, to design anti-GBM cell therapies that use MSCs as vectors

  1. Combination cell therapy with mesenchymal stem cells and neural stem cells for brain stroke in rats.

    Science.gov (United States)

    Hosseini, Seyed Mojtaba; Farahmandnia, Mohammad; Razi, Zahra; Delavari, Somayeh; Shakibajahromi, Benafsheh; Sarvestani, Fatemeh Sabet; Kazemi, Sepehr; Semsar, Maryam

    2015-05-01

    Brain stroke is the second most important events that lead to disability and morbidity these days. Although, stroke is important, there is no treatment for curing this problem. Nowadays, cell therapy has opened a new window for treating central nervous system disease. In some previous studies the Mesenchymal stem cells and neural stem cells. In this study, we have designed an experiment to assess the combination cell therapy (Mesenchymal and Neural stem cells) effects on brain stroke. The Mesenchymal stem cells were isolated from adult rat bone marrow and the neural stem cells were isolated from ganglion eminence of rat embryo 14 days. The Mesenchymal stem cells were injected 1 day after middle cerebral artery occlusion (MCAO) and the neural stem cells transplanted 7 day after MCAO. After 28 days, the neurological outcomes and brain lesion volumes were evaluated. Also, the activity of Caspase 3 was assessed in different groups. The group which received combination cell therapy had better neurological examination and less brain lesion. Also the combination cell therapy group had the least Caspase 3 activity among the groups. The combination cell therapy is more effective than Mesenchymal stem cell therapy and neural stem cell therapy separately in treating the brain stroke in rats.

  2. Acetaldehyde and hexanaldehyde from cultured white cells

    Directory of Open Access Journals (Sweden)

    Zaldivar Frank

    2009-04-01

    Full Text Available Abstract Background Noninvasive detection of innate immune function such as the accumulation of neutrophils remains a challenge in many areas of clinical medicine. We hypothesized that granulocytes could generate volatile organic compounds. Methods To begin to test this, we developed a bioreactor and analytical GC-MS system to accurately identify and quantify gases in trace concentrations (parts per billion emitted solely from cell/media culture. A human promyelocytic leukemia cell line, HL60, frequently used to assess neutrophil function, was grown in serum-free medium. Results HL60 cells released acetaldehyde and hexanaldehyde in a time-dependent manner. The mean ± SD concentration of acetaldehyde in the headspace above the cultured cells following 4-, 24- and 48-h incubation was 157 ± 13 ppbv, 490 ± 99 ppbv, 698 ± 87 ppbv. For hexanaldehyde these values were 1 ± 0.3 ppbv, 8 ± 2 ppbv, and 11 ± 2 ppbv. In addition, our experimental system permitted us to identify confounding trace gas contaminants such as styrene. Conclusion This study demonstrates that human immune cells known to mimic the function of innate immune cells, like neutrophils, produce volatile gases that can be measured in vitro in trace amounts.

  3. Neural Stem Cells in the Diabetic Brain

    Directory of Open Access Journals (Sweden)

    Tomás P. Bachor

    2012-01-01

    Full Text Available Experimental diabetes in rodents rapidly affects the neurogenic niches of the adult brain. Moreover, behavioral disorders suggest that a similar dysfunction of the neurogenic niches most likely affects diabetic and prediabetic patients. Here, we review our present knowledge about adult neural stem cells, the methods used for their study in diabetic models, and the effects of experimental diabetes. Variations in diet and even a short hyperglycemia profoundly change the structure and the proliferative dynamics of the neurogenic niches. Moreover, alterations of diabetic neurogenic niches appear to be associated with diabetic cognitive disorders. Available evidence supports the hypothesis that, in the adult, early changes of the neurogenic niches might enhance development of the diabetic disease.

  4. Long term ex-vivo culturing of Drosophila brain as a method to live image pupal brains: insights into the cellular mechanisms of neuronal remodeling

    Directory of Open Access Journals (Sweden)

    Dana eRabinovich

    2015-08-01

    Full Text Available Holometabolous insects, including Drosophila melanogaster, undergo complete metamorphosis that includes a pupal stage. During metamorphosis, the Drosophila nervous system undergoes massive remodeling and growth, that include cell death and large-scale axon and synapse elimination as well as neurogenesis, developmental axon regrowth and formation of new connections. Neuronal remodeling is an essential step in the development of vertebrate and invertebrate nervous systems. Research on the stereotypic remodeling of Drosophila mushroom body (MB γ neurons has contributed to our knowledge of the molecular mechanisms of remodeling but our knowledge of the cellular mechanisms remain poorly understood. A major hurdle in understanding various dynamic processes that occur during metamorphosis is the lack of time-lapse resolution. The pupal case and opaque fat bodies that enwrap the central nervous system (CNS make live-imaging of the central brain in-vivo impossible. We have established an ex-vivo long-term brain culture system that supports the development and neuronal remodeling of pupal brains. By optimizing culture conditions and dissection protocols, we have observed development in culture at kinetics similar to what occurs in vivo. Using this new method, we have obtained the first time-lapse sequence of MB γ neurons undergoing remodeling in up to a single cell resolution. We found that axon pruning is initiated by blebbing, followed by one-two nicks that seem to initiate a more widely spread axon fragmentation. As such, we have set up some of the tools and methodologies needed for further exploration of the cellular mechanisms of neuronal remodeling, not limited to the MB. The long-term ex-vivo brain culture system that we report here could be used to study dynamic aspects of neurodevelopment of any Drosophila neuron.

  5. Cre Fused with RVG Peptide Mediates Targeted Genome Editing in Mouse Brain Cells In Vivo.

    Science.gov (United States)

    Zou, Zhiyuan; Sun, Zhaolin; Li, Pan; Feng, Tao; Wu, Sen

    2016-12-14

    Cell penetrating peptides (CPPs) are short peptides that can pass through cell membranes. CPPs can facilitate the cellular entry of proteins, macromolecules, nanoparticles and drugs. RVG peptide (RVG hereinafter) is a 29-amino-acid CPP derived from a rabies virus glycoprotein that can cross the blood-brain barrier (BBB) and enter brain cells. However, whether RVG can be used for genome editing in the brain has not been reported. In this work, we combined RVG with Cre recombinase for bacterial expression. The purified RVG-Cre protein cut plasmids in vitro and traversed cell membranes in cultured Neuro2a cells. By tail vein-injecting RVG-Cre into Cre reporter mouse lines mTmG and Rosa26 lacZ , we demonstrated that RVG-Cre could target brain cells and achieve targeted somatic genome editing in adult mice. This direct delivery of the gene-editing enzyme protein into mouse brains with RVG is much safer than plasmid- or viral-based methods, holding promise for further applications in the treatment of various brain diseases.

  6. Mouse cell culture - Methods and protocols

    Directory of Open Access Journals (Sweden)

    CarloAlberto Redi

    2010-12-01

    Full Text Available The mouse is, out of any doubt, the experimental animal par excellence for many many colleagues within the scientific community, notably for those working in mammalian biology (in a broad sense, from basic genetic to modeling human diseases, starting at least from 1664 Robert Hooke experiments on air’s propertyn. Not surprising then that mouse cell cultures is a well established field of research itself and that there are several handbooks devoted to this discipline. Here, Andrew Ward and David Tosh provide a necessary update of the protocols currently needed. In fact, nearly half of the book is devoted to stem cells culture protocols, mainly embryonic, from a list of several organs (kidney, lung, oesophagus and intestine, pancreas and liver to mention some........

  7. Callus and cell suspension cultures of carnation

    DEFF Research Database (Denmark)

    Engvild, Kjeld Christensen

    1972-01-01

    of growth regulators were observed to be 3 × 10−6M indoleacetic acid (JAA) combined with 3 × 10−6M benzylaminopurin (BAP) or 10−6M 2,4-dichlorophenoxy acetic acid (2,4-D) alone. IAA + BAP caused a 100 fold increase in fresh weight over 4 weeks at 25°C. Addition of casein hydrolysate increased growth further....... Cell suspension cultures worked best in media containing 2,4-D in which they had a doubling time of about 2 days. Filtered suspensions were successfully plated on agar in petri dishes, but division was never observed in single cells. The cultures initiated roots at higher concentrations of IAA or NAA...

  8. Conversion of primordial germ cells to pluripotent stem cells: methods for cell tracking and culture conditions.

    Science.gov (United States)

    Nagamatsu, Go; Suda, Toshio

    2013-01-01

    Primordial germ cells (PGCs) are unipotent cells committed to germ lineage: PGCs can only differentiate into gametes in vivo. However, upon fertilization, germ cells acquire the capacity to differentiate into all cell types in the body, including germ cells. Therefore, germ cells are thought to have the potential for pluripotency. PGCs can convert to pluripotent stem cells in vitro when cultured under specific conditions that include bFGF, LIF, and the membrane-bound form of SCF (mSCF). Here, the culture conditions which efficiently convert PGCs to pluripotent embryonic germ (EG) cells are described, as well as methods used for identifying pluripotent candidate cells during culture.

  9. Indirect immunofluorescence staining of cultured neural cells.

    Science.gov (United States)

    Barbierato, Massimo; Argentini, Carla; Skaper, Stephen D

    2012-01-01

    Immunofluorescence is a technique allowing the visualization of a specific protein or antigen in cells or tissue sections by binding a specific antibody chemically conjugated with a fluorescent dye such as fluorescein isothiocyanate. There are two major types of immunofluorescence staining methods: (1) direct immunofluorescence staining in which the primary antibody is labeled with fluorescence dye and (2) indirect immunofluorescence staining in which a secondary antibody labeled with fluorochrome is used to recognize a primary antibody. This chapter describes procedures for the application of indirect immunofluorescence staining to neural cells in culture.

  10. Cultured epidermal stem cells in regenerative medicine.

    Science.gov (United States)

    Jackson, Catherine J; Tønseth, Kim Alexander; Utheim, Tor Paaske

    2017-07-04

    Transplantation of cultured epidermal cell sheets (CES) has long been used to treat patients with burns, chronic wounds, and stable vitiligo. In patients with large area burns this can be a life-saving procedure. The ultimate goal, however, is to restore all normal functions of the skin and prevent scar formation. Increased focus on the incorporation of epidermal stem cells (EpiSCs) within CES transplants may ultimately prove to be key to achieving this. Transplanted EpiSCs contribute to restoring the complete epidermis and provide long-term renewal.Maintenance of the regenerative potential of EpiSCs is anchorage-dependent. The extracellular matrix (ECM) provides physical cues that are interpreted by EpiSCs and reciprocal signaling between cells and ECM are integrated to determine cell fate. Thus, the carrier scaffold chosen for culture and transplant influences maintenance of EpiSC phenotype and may enhance or detract from regenerative healing following transfer.Long-term effectiveness and safety of genetically modified EpiSCs to correct the severe skin blistering disease epidermolysis bullosa has been shown clinically. Furthermore, skin is gaining interest as an easily accessible source of adult epithelial stem cells potentially useful for restoration of other types of epithelia. This review highlights the role of EpiSCs in the current treatment of skin injury and disease, as well as their potential in novel regenerative medicine applications involving other epithelia.

  11. Ascorbic acid transport into cultured pituitary cells

    International Nuclear Information System (INIS)

    Cullen, E.I.; May, V.; Eipper, R.A.

    1986-01-01

    An amidating enzyme designated peptidyl-glycine α-amidating monooxygenase (PAM) has been studied in a variety of tissues and is dependent on molecular oxygen and stimulated by copper and ascorbic acid. To continue investigating the relationship among cellular ascorbic acid concentrations, amidating ability, and PAM activity, the authors studied ascorbic acid transport in three cell preparations that contain PAM and produce amidated peptides: primary cultures of rat anterior and intermediate pituitary and mouse AtT-20 tumor cells. When incubated in 50 μM [ 14 C]ascorbic acid all three cell preparations concentrated ascorbic acid 20- to 40-fold, producing intracellular ascorbate concentrations of 1 to 2 mM, based on experimentally determined cell volumes. All three cell preparations displayed saturable ascorbic acid uptake with half-maximal initial rates occurring between 9 and 18 μM ascorbate. Replacing NaCl in the uptake buffer with choline chloride significantly diminished ascorbate uptake in all three preparations. Ascorbic acid efflux from these cells was slow, displaying half-lives of 7 hours. Unlike systems that transport dehydroascorbic acid, the transport system for ascorbic acid in these cells was not inhibited by glucose. Thus, ascorbate is transported into pituitary cells by a sodium-dependent, active transport system

  12. Cell diversity and network dynamics in photosensitive human brain organoids.

    Science.gov (United States)

    Quadrato, Giorgia; Nguyen, Tuan; Macosko, Evan Z; Sherwood, John L; Min Yang, Sung; Berger, Daniel R; Maria, Natalie; Scholvin, Jorg; Goldman, Melissa; Kinney, Justin P; Boyden, Edward S; Lichtman, Jeff W; Williams, Ziv M; McCarroll, Steven A; Arlotta, Paola

    2017-05-04

    In vitro models of the developing brain such as three-dimensional brain organoids offer an unprecedented opportunity to study aspects of human brain development and disease. However, the cells generated within organoids and the extent to which they recapitulate the regional complexity, cellular diversity and circuit functionality of the brain remain undefined. Here we analyse gene expression in over 80,000 individual cells isolated from 31 human brain organoids. We find that organoids can generate a broad diversity of cells, which are related to endogenous classes, including cells from the cerebral cortex and the retina. Organoids could be developed over extended periods (more than 9 months), allowing for the establishment of relatively mature features, including the formation of dendritic spines and spontaneously active neuronal networks. Finally, neuronal activity within organoids could be controlled using light stimulation of photosensitive cells, which may offer a way to probe the functionality of human neuronal circuits using physiological sensory stimuli.

  13. Ammonium accumulation and cell death in a rat 3D brain cell model of glutaric aciduria type I.

    Directory of Open Access Journals (Sweden)

    Paris Jafari

    Full Text Available Glutaric aciduria type I (glutaryl-CoA dehydrogenase deficiency is an inborn error of metabolism that usually manifests in infancy by an acute encephalopathic crisis and often results in permanent motor handicap. Biochemical hallmarks of this disease are elevated levels of glutarate and 3-hydroxyglutarate in blood and urine. The neuropathology of this disease is still poorly understood, as low lysine diet and carnitine supplementation do not always prevent brain damage, even in early-treated patients. We used a 3D in vitro model of rat organotypic brain cell cultures in aggregates to mimic glutaric aciduria type I by repeated administration of 1 mM glutarate or 3-hydroxyglutarate at two time points representing different developmental stages. Both metabolites were deleterious for the developing brain cells, with 3-hydroxyglutarate being the most toxic metabolite in our model. Astrocytes were the cells most strongly affected by metabolite exposure. In culture medium, we observed an up to 11-fold increase of ammonium in the culture medium with a concomitant decrease of glutamine. We further observed an increase in lactate and a concomitant decrease in glucose. Exposure to 3-hydroxyglutarate led to a significantly increased cell death rate. Thus, we propose a three step model for brain damage in glutaric aciduria type I: (i 3-OHGA causes the death of astrocytes, (ii deficiency of the astrocytic enzyme glutamine synthetase leads to intracerebral ammonium accumulation, and (iii high ammonium triggers secondary death of other brain cells. These unexpected findings need to be further investigated and verified in vivo. They suggest that intracerebral ammonium accumulation might be an important target for the development of more effective treatment strategies to prevent brain damage in patients with glutaric aciduria type I.

  14. Gadolinium uptake by brain cancer cells: Quantitative analysis with X-PEEM spectromicroscopy for cancer therapy

    Science.gov (United States)

    De Stasio, Gelsomina; Gilbert, B.; Perfetti, P.; Margaritondo, G.; Mercanti, D.; Ciotti, M. T.; Casalbore, P.; Larocca, L. M.; Rinelli, A.; Pallini, R.

    2000-05-01

    We present the first X-PEEM spectromicroscopy semi-quantitative data, acquired on Gd in glioblastoma cell cultures from human brain cancer. The cells were treated with a Gd compound for the optimization of GdNCT (Gadolinium Neutron Capture Therapy). We analyzed the kinetics of Gd uptake as a function of exposure time, and verified that a quantitative analytical technique gives the same results as our MEPHISTO X-PEEM, demonstrating the feasibility of semi-quantitative spectromicroscopy.

  15. Obtaining phenolic acids from cell cultures of various Artemisia ...

    African Journals Online (AJOL)

    Plant cell cultures represent a high valuable source for the production of bioactive secondary metabolites which can be used in food industry, medicine and cosmetic industry. In our study, we focused on obtaining phenolic acids from plant cell cultures. We compared cell cultures obtained from nine plant species of two ...

  16. An Introductory Undergraduate Course Covering Animal Cell Culture Techniques

    Science.gov (United States)

    Mozdziak, Paul E.; Petitte, James N.; Carson, Susan D.

    2004-01-01

    Animal cell culture is a core laboratory technique in many molecular biology, developmental biology, and biotechnology laboratories. Cell culture is a relatively old technique that has been sparingly taught at the undergraduate level. The traditional methodology for acquiring cell culture training has been through trial and error, instruction when…

  17. The brain-artefact interface (BAI): a challenge for archaeology and cultural neuroscience.

    Science.gov (United States)

    Malafouris, Lambros

    2010-06-01

    Cultural neuroscience provides a new approach for understanding the impact of culture on the human brain (and vice versa) opening thus new avenues for cross-disciplinary collaboration with archaeology and anthropology. Finding new meaningful and productive unit of analysis is essential for such collaboration. But what can archaeological preoccupation with material culture and long-term change contribute to this end? In this article, I introduce and discuss the notion of the brain-artefact interface (BAI) as a useful conceptual bridge between neuroplastisty and the extended mind. I argue that a key challenge for archaeology and cultural neuroscience lies in the cross-disciplinary understanding of the processes by which our plastic enculturated brains become constituted within the wider extended networks of non-biological artefacts and cultural practices that delineate the real spatial and temporal boundaries of the human cognitive map.

  18. Reversible gelling culture media for in-vitro cell culture in three-dimensional matrices

    Science.gov (United States)

    An, Yuehuei H.; Mironov, Vladimir A.; Gutowska, Anna

    2000-01-01

    A gelling cell culture medium useful for forming a three dimensional matrix for cell culture in vitro is prepared by copolymerizing an acrylamide derivative with a hydrophilic comonomer to form a reversible (preferably thermally reversible) gelling linear random copolymer in the form of a plurality of linear chains having a plurality of molecular weights greater than or equal to a minimum gelling molecular weight cutoff, mixing the copolymer with an aqueous solvent to form a reversible gelling solution and adding a cell culture medium to the gelling solution to form the gelling cell culture medium. Cells such as chondrocytes or hepatocytes are added to the culture medium to form a seeded culture medium, and temperature of the medium is raised to gel the seeded culture medium and form a three dimensional matrix containing the cells. After propagating the cells in the matrix, the cells may be recovered by lowering the temperature to dissolve the matrix and centrifuging.

  19. PHYTOCHEMICAL STUDY OF CELL CULTURE JATROPHA CURCAS

    Directory of Open Access Journals (Sweden)

    KOMAR RUSLAN

    2011-01-01

    Full Text Available Jatropha curcas belongs to the Euphorbiaceae family which has potential economically. This plant has been reported to contain toxic compounds such as curcin and phorbol ester and its derivatives. These compounds may become a problem if J. curcas will be explored as a source of biofuel. In order to provide safety plants, the research on the study of phytochemical and initiation of cell and organ culture have been carried out. J curcas which has been collected from different regions in Indonesia showed to contain relatively the same profile of chemical contents. Dominant compounds that were detected by GCMS are hidrocarbon such as 2-heptenal, decadienal, hexsadecane, pentadecane, cyclooctane etc, fatty acid such as oktadecanoate acid, etthyl linoleate, ethyl stearate, heksadecanoate acid and steroid such as stigmasterol, fucosterol, sitosterol. No phorbol ester and its derivatives have been detected yet by the GCMS method. Callus and suspension cultures of J. curcas have been established to be used for further investigation.

  20. Gold nanoparticle interactions with endothelial cells cultured under physiological conditions.

    Science.gov (United States)

    Freese, C; Anspach, L; Deller, R C; Richards, S-J; Gibson, M I; Kirkpatrick, C J; Unger, R E

    2017-03-28

    PEGylated gold nanoparticles (AuNPs) have an extended circulation time after intravenous injection in vivo and exhibit favorable properties for biosensing, diagnostic imaging, and cancer treatment. No impact of PEGylated AuNPs on the barrier forming properties of endothelial cells (ECs) has been reported, but recent studies demonstrated that unexpected effects on erythrocytes are observed. Almost all studies to date have been with static-cultured ECs. Herein, ECs maintained under physiological cyclic stretch and flow conditions and used to generate a blood-brain barrier model were exposed to 20 nm PEGylated AuNPs. An evaluation of toxic effects, cell stress, the release profile of pro-inflammatory cytokines, and blood-brain barrier properties showed that even under physiological conditions no obvious effects of PEGylated AuNPs on ECs were observed. These findings suggest that 20 nm-sized, PEGylated AuNPs may be a useful tool for biomedical applications, as they do not affect the normal function of healthy ECs after entering the blood stream.

  1. A biocompatible micro cell culture chamber (mu CCC) for the culturing and on-line monitoring of eukaryote cells

    DEFF Research Database (Denmark)

    Stangegaard, Michael; Petronis, Sarunas; Jørgensen, Anders Michael

    2006-01-01

    We have previously shown that a polymeric (PMMA) chip with medium perfusion and integrated heat regulation provides sufficiently precise heat regulation, pH-control and medium exchange to support cell growth for weeks. However, it was unclear how closely the cells cultured in the chip resembled c...... compared to cell cultured in culture flasks incubated in a dark and CO2 conditioned incubator....

  2. Good cell culture practices &in vitro toxicology.

    Science.gov (United States)

    Eskes, Chantra; Boström, Ann-Charlotte; Bowe, Gerhard; Coecke, Sandra; Hartung, Thomas; Hendriks, Giel; Pamies, David; Piton, Alain; Rovida, Costanza

    2017-12-01

    Good Cell Culture Practices (GCCP) is of high relevance to in vitro toxicology. The European Society of Toxicology In Vitro (ESTIV), the Center for Alternatives for Animal Testing (CAAT) and the In Vitro Toxicology Industrial Platform (IVTIP) joined forces to address by means of an ESTIV 2016 pre-congress session the different aspects and applications of GCCP. The covered aspects comprised the current status of the OECD guidance document on Good In Vitro Method Practices, the importance of quality assurance for new technological advances in in vitro toxicology including stem cells, and the optimized implementation of Good Manufacturing Practices and Good Laboratory Practices for regulatory testing purposes. General discussions raised the duality related to the difficulties in implementing GCCP in an academic innovative research framework on one hand, and on the other hand, the need for such GCCP principles in order to ensure reproducibility and robustness of in vitro test methods for toxicity testing. Indeed, if good cell culture principles are critical to take into consideration for all uses of in vitro test methods for toxicity testing, the level of application of such principles may depend on the stage of development of the test method as well as on the applications of the test methods, i.e., academic innovative research vs. regulatory standardized test method. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Adult human brain neural progenitor cells (NPCs and fibroblast-like cells have similar properties in vitro but only NPCs differentiate into neurons.

    Directory of Open Access Journals (Sweden)

    Thomas In-Hyeup Park

    Full Text Available The ability to culture neural progenitor cells from the adult human brain has provided an exciting opportunity to develop and test potential therapies on adult human brain cells. To achieve a reliable and reproducible adult human neural progenitor cell (AhNPC culture system for this purpose, this study fully characterized the cellular composition of the AhNPC cultures, as well as the possible changes to this in vitro system over prolonged culture periods. We isolated cells from the neurogenic subventricular zone/hippocampus (SVZ/HP of the adult human brain and found a heterogeneous culture population comprised of several types of post-mitotic brain cells (neurons, astrocytes, and microglia, and more importantly, two distinct mitotic cell populations; the AhNPCs, and the fibroblast-like cells (FbCs. These two populations can easily be mistaken for a single population of AhNPCs, as they both proliferate under AhNPC culture conditions, form spheres and express neural progenitor cell and early neuronal markers, all of which are characteristics of AhNPCs in vitro. However, despite these similarities under proliferating conditions, under neuronal differentiation conditions, only the AhNPCs differentiated into functional neurons and glia. Furthermore, AhNPCs showed limited proliferative capacity that resulted in their depletion from culture by 5-6 passages, while the FbCs, which appear to be from a neurovascular origin, displayed a greater proliferative capacity and dominated the long-term cultures. This gradual change in cellular composition resulted in a progressive decline in neurogenic potential without the apparent loss of self-renewal in our cultures. These results demonstrate that while AhNPCs and FbCs behave similarly under proliferative conditions, they are two different cell populations. This information is vital for the interpretation and reproducibility of AhNPC experiments and suggests an ideal time frame for conducting Ah

  4. Optogenetic control of human neurons in organotypic brain cultures

    DEFF Research Database (Denmark)

    Andersson, My; Avaliani, Natalia; Svensson, Andreas

    2016-01-01

    Optogenetics is one of the most powerful tools in neuroscience, allowing for selective control of specific neuronal populations in the brain of experimental animals, including mammals. We report, for the first time, the application of optogenetic tools to human brain tissue providing a proof......-of-concept for the use of optogenetics in neuromodulation of human cortical and hippocampal neurons as a possible tool to explore network mechanisms and develop future therapeutic strategies....

  5. Electroporation-based methods for in vivo, whole mount and primary culture analysis of zebrafish brain development

    Directory of Open Access Journals (Sweden)

    Jesuthasan Suresh

    2007-03-01

    Full Text Available Abstract Background Electroporation is a technique for the introduction of nucleic acids and other macromolecules into cells. In chick embryos it has been a particularly powerful technique for the spatial and temporal control of gene expression in developmental studies. Electroporation methods have also been reported for Xenopus, zebrafish, and mouse. Results We present a new protocol for zebrafish brain electroporation. Using a simple set-up with fixed spaced electrodes and microinjection equipment, it is possible to electroporate 50 to 100 embryos in 1 hour with no lethality and consistently high levels of transgene expression in numerous cells. Transfected cells in the zebrafish brain are amenable to in vivo time lapse imaging. Explants containing transfected neurons can be cultured for in vitro analysis. We also present a simple enzymatic method to isolate whole brains from fixed zebrafish for immunocytochemistry. Conclusion Building on previously described methods, we have optimized several parameters to allow for highly efficient unilateral or bilateral transgenesis of a large number of cells in the zebrafish brain. This method is simple and provides consistently high levels of transgenesis for large numbers of embryos.

  6. Electroporation-based methods for in vivo, whole mount and primary culture analysis of zebrafish brain development.

    Science.gov (United States)

    Hendricks, Michael; Jesuthasan, Suresh

    2007-03-15

    Electroporation is a technique for the introduction of nucleic acids and other macromolecules into cells. In chick embryos it has been a particularly powerful technique for the spatial and temporal control of gene expression in developmental studies. Electroporation methods have also been reported for Xenopus, zebrafish, and mouse. We present a new protocol for zebrafish brain electroporation. Using a simple set-up with fixed spaced electrodes and microinjection equipment, it is possible to electroporate 50 to 100 embryos in 1 hour with no lethality and consistently high levels of transgene expression in numerous cells. Transfected cells in the zebrafish brain are amenable to in vivo time lapse imaging. Explants containing transfected neurons can be cultured for in vitro analysis. We also present a simple enzymatic method to isolate whole brains from fixed zebrafish for immunocytochemistry. Building on previously described methods, we have optimized several parameters to allow for highly efficient unilateral or bilateral transgenesis of a large number of cells in the zebrafish brain. This method is simple and provides consistently high levels of transgenesis for large numbers of embryos.

  7. Organotypic hippocampal slice cultures for studies of brain damage, neuroprotection and neurorepair

    DEFF Research Database (Denmark)

    Noraberg, Jens; Poulsen, Frantz Rom; Blaabjerg, Morten

    2005-01-01

    Slices of developing brain tissue can be grown for several weeks as so-called organotypic slice cultures. Here we summarize and review studies using hippocampal slice cultures to investigate mechanisms and treatment strategies for the neurodegenerative disorders like stroke (cerebral ischemia), A...

  8. Brain microvascular pericytes are immunoactive in culture: cytokine, chemokine, nitric oxide, and LRP-1 expression in response to lipopolysaccharide

    Directory of Open Access Journals (Sweden)

    Erickson Michelle A

    2011-10-01

    Full Text Available Abstract Background Brain microvascular pericytes are important constituents of the neurovascular unit. These cells are physically the closest cells to the microvascular endothelial cells in brain capillaries. They significantly contribute to the induction and maintenance of the barrier functions of the blood-brain barrier. However, very little is known about their immune activities or their roles in neuroinflammation. Here, we focused on the immunological profile of brain pericytes in culture in the quiescent and immune-challenged state by studying their production of immune mediators such as nitric oxide (NO, cytokines, and chemokines. We also examined the effects of immune challenge on pericyte expression of low density lipoprotein receptor-related protein-1 (LRP-1, a protein involved in the processing of amyloid precursor protein and the brain-to-blood efflux of amyloid-β peptide. Methods Supernatants were collected from primary cultures of mouse brain pericytes. Release of nitric oxide (NO was measured by the Griess reaction and the level of S-nitrosylation of pericyte proteins measured with a modified "biotin-switch" method. Specific mitogen-activated protein kinase (MAPK pathway inhibitors were used to determine involvement of these pathways on NO production. Cytokines and chemokines were analyzed by multianalyte technology. The expression of both subunits of LRP-1 was analyzed by western blot. Results Lipopolysaccharide (LPS induced release of NO by pericytes in a dose-dependent manner that was mediated through MAPK pathways. Nitrative stress resulted in S-nitrosylation of cellular proteins. Eighteen of twenty-three cytokines measured were released constitutively by pericytes or with stimulation by LPS, including interleukin (IL-12, IL-13, IL-9, IL-10, granulocyte-colony stimulating factor, granulocyte macrophage-colony stimulating factor, eotaxin, chemokine (C-C motif ligand (CCL-3, and CCL-4. Pericyte expressions of both subunits of

  9. A cultural neuroscience approach to the biosocial nature of the human brain.

    Science.gov (United States)

    Han, Shihui; Northoff, Georg; Vogeley, Kai; Wexler, Bruce E; Kitayama, Shinobu; Varnum, Michael E W

    2013-01-01

    Cultural neuroscience (CN) is an interdisciplinary field that investigates the relationship between culture (e.g., value and belief systems and practices shared by groups) and human brain functions. In this review we describe the origin, aims, and methods of CN as well as its conceptual framework and major findings. We also clarify several misunderstandings of CN research. Finally, we discuss the implications of CN findings for understanding human brain function in sociocultural contexts and novel questions that future CN research should address. By doing so, we hope to provide a clear picture of the CN approach to the human brain and culture and to elucidate the intrinsically biosocial nature of the functional organization of the human brain.

  10. Expression of histamine receptor genes Hrh3 and Hrh4 in rat brain endothelial cells.

    Science.gov (United States)

    Karlstedt, K; Jin, C; Panula, P

    2013-09-01

    Brain vascular endothelial cells express histamine H1 and H2 receptors, which regulate brain capillary permeability. We investigated whether H3 and H4 receptors are also expressed in these cells and may thus play a role in permeability regulation. An immortalized rat brain endothelial cell line RBE4 was used to assess the presence of H3 and H4 receptors. Reverse transcription-PCR (RT-PCR) and sequencing were used to identify the receptor mRNAs. The receptors were stimulated with histamine and immepip, and specific inverse agonists/antagonists ciproxifan and JNJ 7777120 were used to block H3 and H4 receptors, respectively. RT-PCR of mRNA extracted from cultured immortalized RBE4 cells revealed two rat H4 receptor gene (Hrh4) transcripts, one full-length (coding sequence 1173 bp), and one with a 164 bp deletion. Also, two rat H3 receptor gene (Hrh3) isoform mRNAs were expressed in RBE4 cells, and sequencing showed they were the full-length H3 receptor and the 144 bp deletion form. Both histamine and immepip (H3 and H4 receptor agonists) activated the Erk1/2 MAPK pathway in the RBE4 cells and in vivo in brain blood vessels by activating H4 receptors, as the H4 receptor-specific inverse agonists/antagonist JNJ 7777120, but not ciproxifan, H3 receptor antagonist, dose-dependently blocked this effect in RBE4 cells. Both Hrh3 and Hrh4 receptors are expressed in rat brain endothelial cells, and activation of the histamine H4 receptor activates the Erk1/2 cascade. H3 and H4 receptors in endothelial cells are potentially important for regulation of blood-brain barrier permeability, including trafficking of immunocompetent cells. © 2013 The Authors. British Journal of Pharmacology © 2013 The British Pharmacological Society.

  11. Enhanced infectivity of bluetongue virus in cell culture by centrifugation.

    OpenAIRE

    Sundin, D R; Mecham, J O

    1989-01-01

    The effects of centrifugation of the infection of cell culture with bluetongue virus (BTV) were investigated. Baby hamster kidney cells were infected with BTV with or without centrifugation. Viral antigen was detected by immunofluorescence at 24 h in both centrifuged and noncentrifuged cultures. However, after 24 h of infection, the production of PFU in centrifuged cell cultures was 10- to 20-fold greater than that seen in cultures not centrifuged. In addition, centrifugation enhanced the dir...

  12. Staphylococcal α-hemolysin is neurotoxic and causes lysis of brain cells in vivo and in vitro.

    Science.gov (United States)

    Dahlberg, Daniel; Mariussen, Espen; Goverud, Ingeborg Løstegaard; Tønjum, Tone; Mæhlen, Jan; Antal, Ellen-Ann; Hassel, Bjørnar

    2015-05-01

    Formation of a bacterial brain abscess entails loss of brain cells and formation of pus. The mechanisms behind the cell loss are not fully understood. Staphylococcus aureus, a common cause of brain abscesses, produces various exotoxins, including α-hemolysin, which is an important factor in brain abscess formation. α-Hemolysin may cause cytolysis by forming pores in the plasma membrane of various eukaryotic cells. However, whether α-hemolysin causes lysis of brain cells is not known. Nor is it known whether α-hemolysin in the brain causes cell death through pore formation or by acting as a chemoattractant, recruiting leukocytes and causing inflammation. Here we show that α-hemolysin injected into rat brain causes cell damage and edema formation within 30 min. Cell damage was accompanied by an increase in extracellular concentrations of zinc, GABA, glutamate, and other amino acids, indicating plasma membrane damage, but leukocytic infiltration was not seen 0.5-12h after α-hemolysin injection. This was in contrast to injection of S. aureus, which triggered extensive infiltration with neutrophils within 8h. In vitro, α-hemolysin caused concentration-dependent lysis of isolated nerve endings and cultured astrocytes. We conclude that α-hemolysin contributes to the cell death inherent in staphylococcal brain abscess formation as a pore-forming neurotoxin. Copyright © 2015 Elsevier Inc. All rights reserved.

  13. Walker 256 tumour cells increase substance P immunoreactivity locally and modify the properties of the blood-brain barrier during extravasation and brain invasion.

    Science.gov (United States)

    Lewis, Kate M; Harford-Wright, Elizabeth; Vink, Robert; Nimmo, Alan J; Ghabriel, Mounir N

    2013-01-01

    It is not yet known how tumour cells traverse the blood-brain barrier (BBB) to form brain metastases. Substance P (SP) release is a key component of neurogenic inflammation which has been recently shown to increase the permeability of the BBB following CNS insults, making it a possible candidate as a mediator of tumour cell extravasation into the brain. This study investigated the properties of the BBB in the early stages of tumour cell invasion into the brain, and the possible involvement of SP. Male Wistar rats were injected with Walker 256 breast carcinoma cells via the internal carotid artery and euthanised at 1, 3, 6 and 9 days post tumour inoculation. Culture medium-injected animals served as controls at 1 and 9 days. Evidence of tumour cell extravasation across the BBB was first observed at 3 days post-inoculation, which corresponded with significantly increased albumin (p p p p < 0.001). The increased SP immunoreactivity and altered BBB properties at 3 days post-inoculation that coincided with early tumour invasion may be indicative of a mechanism for tumour cell extravasation into the brain. Thus, extravasation of tumour cells into the brain to form cerebral metastases may be a SP-mediated process.

  14. [Tumor Cells and Micro-environment in Brain Metastases].

    Science.gov (United States)

    Zhong, Wen; Hu, Chengping

    2016-09-20

    Improvements in survival and quality of life of patients with lung cancer had been achieved due to the progression of early diagnosis and precision medicine at recent years, however, until now, treatments targeted at lesions in central nervous system are far from satisfying, thus threatening livelihood of patients involved. After all, in the issue of prophylaxis and therapeutics of brain metastases, it is crucial to learn about the biological behavior of tumor cells in brain metastases and its mechanism underlying, and the hypothesis "seed and soil", that is, tumor cells would generate series of adaptive changes to fit in the new environment, is liable to help explain this process well. In this assay, we reviewed documents concerning tumor cells, brain micro-environments and their interactions in brain metastases, aiming to provide novel insight into the treatments of brain metastases.

  15. Transmigration of neural stem cells across the blood brain barrier induced by glioma cells.

    Directory of Open Access Journals (Sweden)

    Mónica Díaz-Coránguez

    Full Text Available Transit of human neural stem cells, ReNcell CX, through the blood brain barrier (BBB was evaluated in an in vitro model of BBB and in nude mice. The BBB model was based on rat brain microvascular endothelial cells (RBMECs cultured on Millicell inserts bathed from the basolateral side with conditioned media (CM from astrocytes or glioma C6 cells. Glioma C6 CM induced a significant transendothelial migration of ReNcells CX in comparison to astrocyte CM. The presence in glioma C6 CM of high amounts of HGF, VEGF, zonulin and PGE2, together with the low abundance of EGF, promoted ReNcells CX transmigration. In contrast cytokines IFN-α, TNF-α, IL-12p70, IL-1β, IL-6, IL-8 and IL-10, as well as metalloproteinases -2 and -9 were present in equal amounts in glioma C6 and astrocyte CMs. ReNcells expressed the tight junction proteins occludin and claudins 1, 3 and 4, and the cell adhesion molecule CRTAM, while RBMECs expressed occludin, claudins 1 and 5 and CRTAM. Competing CRTAM mediated adhesion with soluble CRTAM, inhibited ReNcells CX transmigration, and at the sites of transmigration, the expression of occludin and claudin-5 diminished in RBMECs. In nude mice we found that ReNcells CX injected into systemic circulation passed the BBB and reached intracranial gliomas, which overexpressed HGF, VEGF and zonulin/prehaptoglobin 2.

  16. Transmigration of neural stem cells across the blood brain barrier induced by glioma cells.

    Science.gov (United States)

    Díaz-Coránguez, Mónica; Segovia, José; López-Ornelas, Adolfo; Puerta-Guardo, Henry; Ludert, Juan; Chávez, Bibiana; Meraz-Cruz, Noemi; González-Mariscal, Lorenza

    2013-01-01

    Transit of human neural stem cells, ReNcell CX, through the blood brain barrier (BBB) was evaluated in an in vitro model of BBB and in nude mice. The BBB model was based on rat brain microvascular endothelial cells (RBMECs) cultured on Millicell inserts bathed from the basolateral side with conditioned media (CM) from astrocytes or glioma C6 cells. Glioma C6 CM induced a significant transendothelial migration of ReNcells CX in comparison to astrocyte CM. The presence in glioma C6 CM of high amounts of HGF, VEGF, zonulin and PGE2, together with the low abundance of EGF, promoted ReNcells CX transmigration. In contrast cytokines IFN-α, TNF-α, IL-12p70, IL-1β, IL-6, IL-8 and IL-10, as well as metalloproteinases -2 and -9 were present in equal amounts in glioma C6 and astrocyte CMs. ReNcells expressed the tight junction proteins occludin and claudins 1, 3 and 4, and the cell adhesion molecule CRTAM, while RBMECs expressed occludin, claudins 1 and 5 and CRTAM. Competing CRTAM mediated adhesion with soluble CRTAM, inhibited ReNcells CX transmigration, and at the sites of transmigration, the expression of occludin and claudin-5 diminished in RBMECs. In nude mice we found that ReNcells CX injected into systemic circulation passed the BBB and reached intracranial gliomas, which overexpressed HGF, VEGF and zonulin/prehaptoglobin 2.

  17. Growth of cultured porcine retinal pigment epithelial cells

    DEFF Research Database (Denmark)

    Wiencke, A.K.; Kiilgaard, Jens Folke; Nicolini, Jair

    2003-01-01

    To establish and characterize cultures of porcine retinal pigment epithelial (pRPE) cells in order to produce confluent monolayers of cells for transplantation.......To establish and characterize cultures of porcine retinal pigment epithelial (pRPE) cells in order to produce confluent monolayers of cells for transplantation....

  18. Cardiac Cells Beating in Culture: A Laboratory Exercise

    Science.gov (United States)

    Weaver, Debora

    2007-01-01

    This article describes how to establish a primary tissue culture, where cells are taken directly from an organ of a living animal. Cardiac cells are taken from chick embryos and transferred to culture dishes. These cells are not transformed and therefore have a limited life span. However, the unique characteristics of cardiac cells are maintained…

  19. Pericellular oxygen monitoring with integrated sensor chips for reproducible cell culture experiments.

    Science.gov (United States)

    Kieninger, J; Aravindalochanan, K; Sandvik, J A; Pettersen, E O; Urban, G A

    2014-04-01

    Here we present an application, in two tumour cell lines, based on the Sensing Cell Culture Flask system as a cell culture monitoring tool for pericellular oxygen sensing. T-47D (human breast cancer) and T98G (human brain cancer) cells were cultured either in atmospheric air or in a glove-box set at 4% oxygen, in both cases with 5% CO2 in the gas phase. Pericellular oxygen tension was measured with the help of an integrated sensor chip comprising oxygen sensor arrays. Obtained results illustrate variation of pericellular oxygen tension in attached cells covered by stagnant medium. Independent of incubation conditions, low pericellular oxygen concentration levels, usually associated with hypoxia, were found in dense cell cultures. Respiration alone brought pericellular oxygen concentration down to levels which could activate hypoxia-sensing regulatory processes in cultures believed to be aerobic. Cells in culture believed to experience conditions of mild hypoxia may, in reality, experience severe hypoxia. This would lead to incorrect assumptions and suggests that pericellular oxygen concentration readings are of great importance to obtain reproducible results when dealing with hypoxic and normoxic (aerobic) incubation conditions. The Sensing Cell Culture Flask system allows continuous monitoring of pericellular oxygen concentration with outstanding long-term stability and no need for recalibration during cell culture experiments. The sensor is integrated into the flask bottom, thus in direct contact with attached cells. No additional equipment needs to be inserted into the flask during culturing. Transparency of the electrochemical sensor chip allows optical inspection of cells attached on top of the sensor. © 2014 John Wiley & Sons Ltd.

  20. Equipment for large-scale mammalian cell culture.

    Science.gov (United States)

    Ozturk, Sadettin S

    2014-01-01

    This chapter provides information on commonly used equipment in industrial mammalian cell culture, with an emphasis on bioreactors. The actual equipment used in the cell culture process can vary from one company to another, but the main steps remain the same. The process involves expansion of cells in seed train and inoculation train processes followed by cultivation of cells in a production bioreactor. Process and equipment options for each stage of the cell culture process are introduced and examples are provided. Finally, the use of disposables during seed train and cell culture production is discussed.

  1. Value of brain computed tomography in small cell lung cancers

    International Nuclear Information System (INIS)

    Fernet, M.; Breau, J.L.; Goldlust, D.; Israel, L.

    1988-01-01

    88 patients with small cell lung cancer were studied. Brain scans were performed first at initial staging and repeated at regular intervals during the survey. The results confirm the limited value of brain scans in the detection of metastases in neurologically asymptomatic patients [fr

  2. [Research progress of cell co-culture method].

    Science.gov (United States)

    Qin, Yanqin; Chen, Yulong; Li, Jiansheng

    2016-08-01

    Cell culture technology is the most commonly used method in the in vitro experiments at present. However, monolayer cell culture technology has been unable to meet the demand of the researchers. This is because that monolayer cell culture cannot mimic the cellular environment in which multiple cells interact with each other in the body. We cannot discuss the relationship of many cells, because we do not know the relationship between cells through a single kind of cell. So cell co-culture medicine arises at the historic moment for the demand. With the development of research method in recent years, cell co-culture method also has been improved in practice: from direct contact co-cultures to indirect contact co-cultures, from two-dimensional co-cultures to three-dimensional co-cultures. Cell co-culture method is closer to the human body. It is also more advantageous to study the interaction among cells. Nowadays, there are more researchers tend to select this method to study the physiological and pathological in vitro model, tissue engineering, and cell differentiation research. At the same time, it has become the focus of drug research and development, drug analysis, mechanism of drug action, and drug targets. This article will review the studies of cell co-culture method, summarize advantages and disadvantages of various methods, so as to promote improvement of cell culture methods, to build cells co-culture system that more close to human body, and build the in vitro model that simulate internal circulation of human body further.

  3. Propagation of human germ stem cells in long-term culture

    Science.gov (United States)

    Akhondi, Mohammad Mehdi; Mohazzab, Arash; Jeddi-Tehrani, Mahmood; Sadeghi, Mohammad Reza; Eidi, Akram; Khodadadi, Abbas; Piravar, Zeinab

    2013-01-01

    Background: Spermatogonial stem cells (SSCs), a subset of undifferentiated type A spermatogonia, are the foundation of complex process of spermatogenesis and could be propagated in vitro culture conditions for long time for germ cell transplantation and fertility preservation. Objective: The aim of this study was in vitro propagation of human spermatogonial stem cells (SSCs) and improvement of presence of human Germ Stem Cells (hGSCs) were assessed by specific markers POU domain, class 5, transcription factor 1 (POU5F1), also known as Octamer-binding transcription factor 4 (Oct-4) and PLZF (Promyelocytic leukaemia zinc finger protein). Materials and Methods: Human testicular cells were isolated by enzymatic digestion (Collagenase IV and Trypsin). Germ cells were cultured in Stem-Pro 34 media supplemented by growth factors such as glial cell line-derived neurotrophic factor, basic fibroblast growth factor, epidermal growth factor and leukemia inhibitory factor to support self-renewal divisions. Germline stem cell clusters were passaged and expanded every week. Immunofluorecent study was accomplished by Anti-Oct4 antibody through the culture. The spermatogonial stem cells genes expression, PLZF, was studied in testis tissue and germ stem cells entire the culture. Results: hGSCs clusters from a brain dead patient developed in testicular cell culture and then cultured and propagated up to 6 weeks. During the culture Oct4 were a specific marker for identification of hGSCs in testis tissue. Expression of PLZF was applied on RNA level in germ stem cells. Conclusion: hGSCs indicated by SSCs specific marker can be cultured and propagated for long-term in vitro conditions. This article extracted from Ph.D. Thesis. (Zeinab Piravar) PMID:24639790

  4. Protection of cultured mammalian cells by rebamipide

    Energy Technology Data Exchange (ETDEWEB)

    Antoku, Shigetoshi; Aramaki, Ryoji [Kyushu Univ., Fukuoka (Japan). Faculty of Medicine; Tanaka, Hisashi; Kusumoto, Naotoshi

    1997-06-01

    Rebamipide which is used as a drug for gastritis and stomach ulcer has large capability for OH radical scavenging. It is expected that rebamipide has protective effect against ionizing radiations. The present paper deals with protective effect of rebamipide for cultured mammalian cells exposed to ionizing radiations. As rebamipide is insoluble in water, three solvents were used to dissolve. Rebamipide dissolved in dimethyl sulfoxide (DMSO), dimethyl formamide (DMFA) and 0.02 N NaOH was added to the cells in Eagle`s minimum essential medium (MEM) supplemented with 10% fetal calf serum and the cells were irradiated with X-rays. After irradiation, the cells were trypsinized, plated in MEM with 10% fetal calf serum and incubated for 7 days in a CO{sub 2} incubator to form colonies. Rebamipide dissolved in 0.02 N NaOH exhibited the protective effect expected its OH radical scavenging capability. However, the protective effect of rebamipide dissolved in DMSO was about half of that expected by its radical scavenging capability and that of rebamipide dissolved in DMFA was not observed. Uptake of rebamipide labeled with {sup 14}C increased with increasing contact time with rebamipide. These rebamipide mainly distributed in nucleus rather than cytoplasm. (author)

  5. Recombinant Protein Production and Insect Cell Culture and Process

    Science.gov (United States)

    Spaulding, Glenn F. (Inventor); Goodwin, Thomas J. (Inventor); OConnor, Kim C. (Inventor); Francis, Karen M. (Inventor); Andrews, Angela D. (Inventor); Prewett, Tracey L. (Inventor)

    1997-01-01

    A process has been developed for recombinant production of selected polypeptides using transformed insect cells cultured in a horizontally rotating culture vessel modulated to create low shear conditions. A metabolically transformed insect cell line is produced using the culture procedure regardless of genetic transformation. The recombinant polypeptide can be produced by an alternative process using virtually infected or stably transformed insect cells containing a gene encoding the described polypeptide. The insect cells can also be a host for viral production.

  6. Modeling chronic brain exposure to amphetamines using primary rat neuronal cortical cultures.

    Science.gov (United States)

    Nogueira, T B; da Costa Araújo, S; Carvalho, F; Pereira, F C; Fernandes, E; Bastos, M L; Costa, V M; Capela, J P

    2014-09-26

    Amphetamine-type psychostimulants (ATS) are used worldwide by millions of patients for several psychiatric disorders. Amphetamine (AMPH) and "ecstasy" (3,4-methylenedioxymethamphetamine or MDMA) are common drugs of abuse. The impact of chronic ATS exposure to neurons and brain aging is still undisclosed. Current neuronal culture paradigms are designed to access acute ATS toxicity. We report for the first time a model of chronic exposure to AMPH and MDMA using long-term rat cortical cultures. In two paradigms, ATS were applied to neurons at day 1 in vitro (DIV) (0, 1, 10 and 100 μM of each drug) up to 28 days (200 μM was applied to cultures up to 14 DIV). Our reincubation protocol assured no decrease in the neuronal media's drug concentration. Chronic exposure of neurons to concentrations equal to or above 100 μM of ATS up to 28 DIV promoted significant mitochondrial dysfunction and elicited neuronal death, which was not prevented by glutamate receptor antagonists at 14 DIV. ATS failed to promote accelerated senescence as no increase in β-galactosidase activity at 21 DIV was found. In younger cultures (4 or 8 DIV), AMPH promoted mitochondrial dysfunction and neuronal death earlier than MDMA. Overall, AMPH proved more toxic and was the only drug that decreased intraneuronal glutathione levels. Meanwhile, caspase 3 activity increased for either drug at 200 μM in younger cultures at 8 DIV, but not at 14 DIV. At 8 DIV, ATS promoted a significant change in the percentage of neurons and astroglia present in culture, promoting a global decrease in the number of both cells. Importantly, concentrations equal to or below 10 μM of either drug did not promote neuronal death or oxidative stress. Our paradigm of neuronal cultures long-term exposure to low micromolar concentrations of ATS closely reproduces the in vivo scenario, being valuable to study the chronic impact of ATS. Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.

  7. Radiosensitivity and TP 53, EGFR amplification and LOH10 analysis of primary glioma cell cultures

    International Nuclear Information System (INIS)

    Gerlach, B.; Harder, A.H.; Slotman, B.J.; Sminia, P.; Hulsebos, T.J.M.; Leenstra, S.; Peter Vandertop, W.; Hartmann, K.A.

    2002-01-01

    Aim: Determination of in-vitro radiosensitivity and genetic alterations of cell cultures derived from human glioma biopsy tissue and established glioma cell lines. Material and Methods: Fresh brain tumor specimens of six patients were processed to early passage cell cultures. In addition the cell lines D 384 and Gli 6 were used. Cell cultures were irradiated with doses from 2 to 10 Gy. Following irradiation, cell survival was determined by clonogenic assay and survival curves were generated. The surviving fractions after 2 Gy (SF2) and 4 Gy (SF4) were used as radiosensitivity parameters. Genetic analysis included determination of the mutational and loss of heterozygosity (LOH) status of TP 53 (exons 5-8), the LOH 10- and epidermal growth factor receptor gene (EGFR) amplification status. Results: The SF2 and SF4 values ranged from 0.54 to 0.88 (mean: 0.70) and from 0.13 to 0.52 (mean: 0.32), respectively. Genetic alterations were found in the Gli 6 cell line and in two primary cell cultures. The genetic profile of Gli 6 showed LOH but no TP 53 mutation, complete LOH 10 and no EGFR amplification. The VU 15 cell culture showed TP 53 mutation but no LOH 10 or EGFR amplification, while VU 24 showed incomplete LOH 10, EGFR amplification and no TP 53 mutation. In the other four cell cultures and D 384 cell line no genetic alterations were diagnosed. Histopathological classification of glioblastoma multiforme and/or genetic alterations resulted in lower radiosensitivity. Conclusion: In this small series of early passage glioma cell cultures low radiosensitivity and alterations in cell regulatory genes were seen. Further testing of biological behavior in larger series of patient-derived material is ongoing. (orig.)

  8. NMR imaging of cell phone radiation absorption in brain tissue

    Science.gov (United States)

    Gultekin, David H.; Moeller, Lothar

    2013-01-01

    A method is described for measuring absorbed electromagnetic energy radiated from cell phone antennae into ex vivo brain tissue. NMR images the 3D thermal dynamics inside ex vivo bovine brain tissue and equivalent gel under exposure to power and irradiation time-varying radio frequency (RF) fields. The absorbed RF energy in brain tissue converts into Joule heat and affects the nuclear magnetic shielding and the Larmor precession. The resultant temperature increase is measured by the resonance frequency shift of hydrogen protons in brain tissue. This proposed application of NMR thermometry offers sufficient spatial and temporal resolution to characterize the hot spots from absorbed cell phone radiation in aqueous media and biological tissues. Specific absorption rate measurements averaged over 1 mg and 10 s in the brain tissue cover the total absorption volume. Reference measurements with fiber optic temperature sensors confirm the accuracy of the NMR thermometry. PMID:23248293

  9. Gravity, chromosomes, and organized development in aseptically cultured plant cells

    Science.gov (United States)

    Krikorian, Abraham D.

    1993-01-01

    The objectives of the PCR experiment are: to test the hypothesis that microgravity will in fact affect the pattern and developmental progression of embryogenically competent plant cells from one well-defined, critical stage to another; to determine the effects of microgravity in growth and differentiation of embryogenic carrot cells grown in cell culture; to determine whether microgravity or the space environment fosters an instability of the differentiated state; and to determine whether mitosis and chromosome behavior are adversely affected by microgravity. The methods employed will consist of the following: special embryogenically competent carrot cell cultures will be grown in cell culture chambers provided by NASDA; four cell culture chambers will be used to grow cells in liquid medium; two dishes (plant cell culture dishes) will be used to grow cells on a semi-solid agar support; progression to later embryonic stages will be induced in space via crew intervention and by media manipulation in the case of liquid grown cell cultures; progression to later stages in case of semi-solid cultures will not need crew intervention; embryo stages will be fixed at a specific interval (day 6) in flight only in the case of liquid-grown cultures; and some living cells and somatic embryos will be returned for continued post-flight development and 'grown-out.' These will derive from the semi-solid grown cultures.

  10. The anti-apoptotic effect of fluid mechanics preconditioning by cells membrane and mitochondria in rats brain microvascular endothelial cells.

    Science.gov (United States)

    Tian, Shan; Zhu, Fengping; Hu, Ruiping; Tian, Song; Chen, Xingxing; Lou, Dan; Cao, Bing; Chen, Qiulei; Li, Bai; Li, Fang; Bai, Yulong; Wu, Yi; Zhu, Yulian

    2018-01-01

    Exercise preconditioning is a simple and effective way to prevent ischemia. This paper further provided the mechanism in hemodynamic aspects at the cellular level. To study the anti-apoptotic effects of fluid mechanics preconditioning, Cultured rats brain microvascular endothelial cells were given fluid intervention in a parallel plate flow chamber before oxygen glucose deprivation. It showed that fluid mechanics preconditioning could inhibit the apoptosis of endothelial cells, and this process might be mediated by the shear stress activation of Tie-2 on cells membrane surface and Bcl-2 on the mitochondria surface. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Copine1 regulates neural stem cell functions during brain development.

    Science.gov (United States)

    Kim, Tae Hwan; Sung, Soo-Eun; Cheal Yoo, Jae; Park, Jae-Yong; Yi, Gwan-Su; Heo, Jun Young; Lee, Jae-Ran; Kim, Nam-Soon; Lee, Da Yong

    2018-01-01

    Copine 1 (CPNE1) is a well-known phospholipid binding protein in plasma membrane of various cell types. In brain cells, CPNE1 is closely associated with AKT signaling pathway, which is important for neural stem cell (NSC) functions during brain development. Here, we investigated the role of CPNE1 in the regulation of brain NSC functions during brain development and determined its underlying mechanism. In this study, abundant expression of CPNE1 was observed in neural lineage cells including NSCs and immature neurons in human. With mouse brain tissues in various developmental stages, we found that CPNE1 expression was higher at early embryonic stages compared to postnatal and adult stages. To model developing brain in vitro, we used primary NSCs derived from mouse embryonic hippocampus. Our in vitro study shows decreased proliferation and multi-lineage differentiation potential in CPNE1 deficient NSCs. Finally, we found that the deficiency of CPNE1 downregulated mTOR signaling in embryonic NSCs. These data demonstrate that CPNE1 plays a key role in the regulation of NSC functions through the activation of AKT-mTOR signaling pathway during brain development. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. Edaravone protects against methylglyoxal-induced barrier damage in human brain endothelial cells.

    Directory of Open Access Journals (Sweden)

    Andrea E Tóth

    Full Text Available Elevated level of reactive carbonyl species, such as methylglyoxal, triggers carbonyl stress and activates a series of inflammatory responses leading to accelerated vascular damage. Edaravone is the active substance of a Japanese medicine, which aids neurological recovery following acute brain ischemia and subsequent cerebral infarction. Our aim was to test whether edaravone can exert a protective effect on the barrier properties of human brain endothelial cells (hCMEC/D3 cell line treated with methylglyoxal.Cell viability was monitored in real-time by impedance-based cell electronic sensing. The barrier function of the monolayer was characterized by measurement of resistance and flux of permeability markers, and visualized by immunohistochemistry for claudin-5 and β-catenin. Cell morphology was also examined by holographic phase imaging.Methylglyoxal exerted a time- and dose-dependent toxicity on cultured human brain endothelial cells: a concentration of 600 µM resulted in about 50% toxicity, significantly reduced the integrity and increased the permeability of the barrier. The cell morphology also changed dramatically: the area of cells decreased, their optical height significantly increased. Edaravone (3 mM provided a complete protection against the toxic effect of methylglyoxal. Co-administration of edaravone restored cell viability, barrier integrity and functions of brain endothelial cells. Similar protection was obtained by the well-known antiglycating molecule, aminoguanidine, our reference compound.These results indicate for the first time that edaravone is protective in carbonyl stress induced barrier damage. Our data may contribute to the development of compounds to treat brain endothelial dysfunction in carbonyl stress related diseases.

  13. X-ray microanalysis of single and cultured cells

    International Nuclear Information System (INIS)

    Wroblewski, J.; Roomans, G.M.

    1984-01-01

    X-ray microanalysis of single or cultured cells is often a useful alternative or complement to the analysis of the corresponding tissue. It also allows the analysis of individual cells in a cell population. Preparation for X-ray microanalysis poses a number of typical problems. Suspensions of single cells can be prepared by either of two pathways: (1) washing - mounting - drying, or (2) centrifugation - freezing or fixation - sectioning. The washing step in the preparation of single or cultured cells presents the most severe problems. Cultured cells are generally grown on a substrate that is compatible with both the analysis and the culture, washed and dried. In some cases, sectioning of cultured cell monolayers has been performed. Special problems in quantitative analysis occur in those cases where the cells are analyzed on a thick substrate, since the substrate contributes to the spectral background

  14. Cholera toxin stimulation of human mammary epithelial cells in culture

    Energy Technology Data Exchange (ETDEWEB)

    Stampfer, M.R.

    1982-06-01

    Addition of cholera toxin to human mammary epithelial cultures derived from reduction mammoplasties and primary carcinomas greatly stimulated cell growth and increased the number of times the cells could be successfully subcultured. Other agents known to increase intracellular cAMP levels were also growth stimulatory. The increased growth potential conferred by cholera toxin enhances the usefulness of this cell culture system.

  15. Establishment and characterization of American elm cell suspension cultures

    Science.gov (United States)

    Steven M. Eshita; Joseph C. Kamalay; Vicki M. Gingas; Daniel A. Yaussy

    2000-01-01

    Cell suspension cultures of Dutch elm disease (DED)-tolerant and DED-susceptible American elms clones have been established and characterized as prerequisites for contrasts of cellular responses to pathogen-derived elicitors. Characteristics of cultured elm cell growth were monitored by A700 and media conductivity. Combined cell growth data for all experiments within a...

  16. Programmed Necrosis: A Prominent Mechanism of Cell Death following Neonatal Brain Injury

    Directory of Open Access Journals (Sweden)

    Raul Chavez-Valdez

    2012-01-01

    Full Text Available Despite the introduction of therapeutic hypothermia, neonatal hypoxic ischemic (HI brain injury remains a common cause of developmental disability. Development of rational adjuvant therapies to hypothermia requires understanding of the pathways of cell death and survival modulated by HI. The conceptualization of the apoptosis-necrosis “continuum” in neonatal brain injury predicts mechanistic interactions between cell death and hydrid forms of cell death such as programmed or regulated necrosis. Many of the components of the signaling pathway regulating programmed necrosis have been studied previously in models of neonatal HI. In some of these investigations, they participate as part of the apoptotic pathways demonstrating clear overlap of programmed death pathways. Receptor interacting protein (RIP-1 is at the crossroads between types of cellular death and survival and RIP-1 kinase activity triggers formation of the necrosome (in complex with RIP-3 leading to programmed necrosis. Neuroprotection afforded by the blockade of RIP-1 kinase following neonatal HI suggests a role for programmed necrosis in the HI injury to the developing brain. Here, we briefly review the state of the knowledge about the mechanisms behind programmed necrosis in neonatal brain injury recognizing that a significant proportion of these data derive from experiments in cultured cell and some from in vivo adult animal models. There are still more questions than answers, yet the fascinating new perspectives provided by the understanding of programmed necrosis in the developing brain may lay the foundation for new therapies for neonatal HI.

  17. High-throughput single-cell manipulation in brain tissue.

    Directory of Open Access Journals (Sweden)

    Joseph D Steinmeyer

    Full Text Available The complexity of neurons and neuronal circuits in brain tissue requires the genetic manipulation, labeling, and tracking of single cells. However, current methods for manipulating cells in brain tissue are limited to either bulk techniques, lacking single-cell accuracy, or manual methods that provide single-cell accuracy but at significantly lower throughputs and repeatability. Here, we demonstrate high-throughput, efficient, reliable, and combinatorial delivery of multiple genetic vectors and reagents into targeted cells within the same tissue sample with single-cell accuracy. Our system automatically loads nanoliter-scale volumes of reagents into a micropipette from multiwell plates, targets and transfects single cells in brain tissues using a robust electroporation technique, and finally preps the micropipette by automated cleaning for repeating the transfection cycle. We demonstrate multi-colored labeling of adjacent cells, both in organotypic and acute slices, and transfection of plasmids encoding different protein isoforms into neurons within the same brain tissue for analysis of their effects on linear dendritic spine density. Our platform could also be used to rapidly deliver, both ex vivo and in vivo, a variety of genetic vectors, including optogenetic and cell-type specific agents, as well as fast-acting reagents such as labeling dyes, calcium sensors, and voltage sensors to manipulate and track neuronal circuit activity at single-cell resolution.

  18. Electrospinning of microbial polyester for cell culture

    International Nuclear Information System (INIS)

    Kwon, Oh Hyeong; Lee, Ik Sang; Ko, Young-Gwang; Meng, Wan; Jung, Kyung-Hye; Kang, Inn-Kyu; Ito, Yoshihiro

    2007-01-01

    Biodegradable and biocompatible poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), a copolymer of microbial polyester, was fabricated as a nanofibrous mat by electrospinning. The specific surface area and the porosity of electrospun PHBV nanofibrous mat were determined. When the mechanical properties of flat film and electrospun PHBV nanofibrous mats were investigated, both the tensile modulus and strength of electrospun PHBV were less than those of cast PHBV film. However, the elongation ratio of nanofiber mat was higher than that of the cast film. The structure of electrospun nanofibers using PHBV-trifluoroethanol solutions depended on the solution concentrations. When x-ray diffraction patterns of bulk PHBV before and after electrospinning were compared, the crystallinity of PHBV was not significantly affected by the electrospinning process. Chondrocytes adhered and grew on the electrospun PHBV nanofibrous mat better than on the cast PHBV film. Therefore, the electrospun PHBV was considered to be suitable for cell culture

  19. Initial Attempts of Development and Characterization of an In Vitro Blood Brain Barrier Model Derived from Human Pluripotent Stem Cells

    DEFF Research Database (Denmark)

    Goldeman, Charlotte; Saaby, Lasse; Hall, Vanessa Jane

    The human blood brain barrier has yet to be successfully replicated as an in vitro model. One of the more promising approaches has been to develop an in vitro model derived from human pluripotent stem cells. However, as promising as this model may be, a successful replication of the differentiation...... method on different kinds of pluripotent stem cell lines have yet to be accomplished. We try to approach the promising method as described by Stebbins et al. (2015) to differentiate human pluripotent stem cells into brain like endothelial cells (BECs). Five different human pluripotent stem cell lines...... were screened for the possibility to differentiate into BECs. Tüb1159, Tüb16423, Bioni010-C, WTSli024-A and WTSli002-A stem cell lines were initially seeded on Matrigel cultured with mTESR1 media to confluence, then seeded on Matrigel as a single cell suspension. After two-three days of culture we...

  20. Usability and Applicability of Microfluidic Cell Culture Systems

    DEFF Research Database (Denmark)

    Hemmingsen, Mette

    of the microfluidic perfusion cell culture system is shown by investigation of adipose-derived stem cell (ASC) differentiation into adipocytes, where we have revealed that paracrine/autocrine signaling is involved in differentiation of a population of ASCs into adipocytes. We have thereby demonstrated......Microfluidic cell culture has been a research area with great attention the last decade due to its potential to mimic the in vivo cellular environment more closely compared to what is possible by conventional cell culture methods. Many exciting and complex devices have been presented providing...... possibilities for, for example, precise control of the chemical environment, 3D cultures, controlled co-culture of different cell types or automated, individual control of up to 96 cell culture chambers in one integrated system. Despite the great new opportunities to perform novel experimental designs...

  1. Can established cultured papilloma cells harbor bovine papillomavirus?

    Science.gov (United States)

    Campos, S R C; Trindade, C; Ferraz, O P; Giovanni, D N S; Lima, A A; Caetano, H V A; Carvalho, R F; Birgel, E H; Dagli, M L Z; Mori, E; Brandão, P E; Richtzenhain, L J; Beçak, W; Stocco, R C

    2008-10-21

    Papillomaviruses have been reported to be very difficult to grow in cell culture. Also, there are no descriptions of cell cultures from lesions of bovine cutaneous papillomatosis, with identification of different bovine papilloma virus (BPV) DNA sequences. In the present report, we describe primary cell cultures from samples of cutaneous lesions (warts). We investigated the simultaneous presence of different BPV DNA sequences, comparing the original lesion to different passages of the cell cultures and to peripheral blood. BPV 1, 2 and 4 DNA sequences were found in lesion samples, and respective cell cultures and peripheral blood, supporting our previous hypothesis of the possible activity of these sequences in different samples and now also showing how they can be maintained in different passages of cell cultures.

  2. Scaling up: Material culture as scaffold for the social brain

    OpenAIRE

    Coward, Fiona

    2016-01-01

    Many other species besides Homo sapiens are tool-users and even tool-makers, but one aspect of material culture still sets modern humans apart: our emotional and social engagement with objects. Here I argue that this engagement acted as a crucial scaffold for the scaling-up of human social networks beyond those of our closest relatives the chimpanzees to the global ‘small world’ of modern humans. Material culture plays a crucial role in conveying social information about relationships between...

  3. Application of cell sheet technology to bone marrow stromal cell transplantation for rat brain infarct.

    Science.gov (United States)

    Ito, Masaki; Shichinohe, Hideo; Houkin, Kiyohiro; Kuroda, Satoshi

    2017-02-01

    Bone marrow stromal cells (BMSC) transplantation enhances functional recovery after cerebral infarct, but the optimal delivery route is undetermined. This study was aimed to assess whether a novel cell-sheet technology non-invasively serves therapeutic benefits to ischemic stroke. First, the monolayered cell sheet was engineered by culturing rat BMSCs on a temperature-responsive dish. The cell sheet was analysed histologically and then transplanted onto the ipsilateral neocortex of rats subjected to permanent middle cerebral artery occlusion at 7 days after the insult. Their behaviours and histology were compared with those in the animals treated with direct injection of BMSCs or vehicle over 4 weeks post-transplantation. The cell sheet was 27.9 ± 8.0 μm thick and was composed of 9.8 ± 2.4 × 10 5 cells. Cell sheet transplantation significantly improved motor function when compared with the vehicle-injected animals. Histological analysis revealed that the BMSCs were densely distributed to the neocortex adjacent to the cerebral infarct and expressed neuronal phenotype in the cell sheet-transplanted animals. These findings were almost equal to those for the animals treated with direct BMSC injection. The attachment of the BMSC sheet to the brain surface did not induce reactive astrocytes in the adjacent neocortex, although direct injection of BMSCs profoundly induced reactive astrocytes around the injection site. These findings suggest that the BMSCs in cell sheets preserve their biological capacity of migration and neural differentiation. Cell-sheet technology may enhance functional recovery after ischaemic stroke, using a less invasive method. Copyright © 2014 John Wiley & Sons, Ltd. Copyright © 2014 John Wiley & Sons, Ltd.

  4. Three-dimensional hydrogel cell culture systems for modeling neural tissue

    Science.gov (United States)

    Frampton, John

    Two-dimensional (2-D) neural cell culture systems have served as physiological models for understanding the cellular and molecular events that underlie responses to physical and chemical stimuli, control sensory and motor function, and lead to the development of neurological diseases. However, the development of three-dimensional (3-D) cell culture systems will be essential for the advancement of experimental research in a variety of fields including tissue engineering, chemical transport and delivery, cell growth, and cell-cell communication. In 3-D cell culture, cells are provided with an environment similar to tissue, in which they are surrounded on all sides by other cells, structural molecules and adhesion ligands. Cells grown in 3-D culture systems display morphologies and functions more similar to those observed in vivo, and can be cultured in such a way as to recapitulate the structural organization and biological properties of tissue. This thesis describes a hydrogel-based culture system, capable of supporting the growth and function of several neural cell types in 3-D. Alginate hydrogels were characterized in terms of their biomechanical and biochemical properties and were functionalized by covalent attachment of whole proteins and peptide epitopes. Methods were developed for rapid cross-linking of alginate hydrogels, thus permitting the incorporation of cells into 3-D scaffolds without adversely affecting cell viability or function. A variety of neural cell types were tested including astrocytes, microglia, and neurons. Cells remained viable and functional for longer than two weeks in culture and displayed process outgrowth in 3-D. Cell constructs were created that varied in cell density, type and organization, providing experimental flexibility for studying cell interactions and behavior. In one set of experiments, 3-D glial-endothelial cell co-cultures were used to model blood-brain barrier (BBB) structure and function. This co-culture system was

  5. Brain fag syndrome: a culture-bound syndrome that may be approaching extinction.

    Science.gov (United States)

    Ayonrinde, Oyedeji A; Obuaya, Chiedu; Adeyemi, Solomon Olusola

    2015-08-01

    Aims and method To explore the current salience of 'brain fag' as a nosological, diagnostic and clinical construct in modern West African psychiatry. A semi-structured questionnaire and vignette based on classical symptoms of brain fag syndrome were used to explore current knowledge, explanatory models and practice among Nigerian psychiatrists. Results Of 102 psychiatrists who responded, 98% recognised the term 'brain fag syndrome' and most recognised the scenario presented. However, only 22% made a diagnosis of brain fag syndrome in their practice preferring diagnoses of anxiety, affective and somatic disorders. Clinical implications A decreasing number of Nigerian psychiatrists are making a diagnosis of 'brain fag syndrome'. We found strong evidence of nosological and diagnostic decline in the syndrome in its place of birth. This may signal the early extinction of this disorder or nosological metamorphosis from a 'culture-bound' syndrome in West African psychiatric practice.

  6. [Effects on proliferation ability of vascular smooth muscle cells by static and/or dynamic cell culture: utility of pre-seeding technique for dynamic cell culture].

    Science.gov (United States)

    Yokomuro, Hiroki; Ozawa, Tsukasa; Fujii, Takeshiro; Shiono, Noritsugu; Watanabe, Yoshinori; Yoshihara, Katsunori; Koyama, Nobuya; Okada, Mitsumasa

    2007-11-01

    Conventional biomaterials are not viable, do not grow, and do not provide contractile effects in cardiac tissue. Foreign synthetic material may become thrombogenic or infected. The most recent cardiac constructs consist of biodegradable material which has the potential to solve these problems. However, dynamic three-dimensional cell culture is necessary because conventional culture is limited to construct tough biografts. Vascular smooth muscle cells derived from rat aorta were seeded to poly-L-lactide-epsilon-capro-lactone copolymer in three groups; static culture group (static cell seeding + static cell culture), dynamic culture group (dynamic cell seeding + dynamic cell culture), and pre-seeding group [static cell seeding and culture for 1 week (pre-seeding) + dynamic cell culture]. The dynamic cell culture system used an original spinner flask. The pre-seeding technique used static cell seeding and culture before dynamic culture. The three groups were evaluated by cell proliferation and histologic studies. Vascular smooth muscle cells could be proliferated in/on the biodegradable materials. The pre-seeding group cells grew much more efficiently than the other groups. Very few cells were found in the biodegradable materials with the dynamic groups. However, there were many cells in the materials with the static culture group and pre-seeding group, especially the pre-seeding group. Dynamic culture is useful for constructing tough biografts by the pre-seeding technique.

  7. Live-cell imaging to detect phosphatidylserine externalization in brain endothelial cells exposed to ionizing radiation: implications for the treatment of brain arteriovenous malformations.

    Science.gov (United States)

    Zhao, Zhenjun; Johnson, Michael S; Chen, Biyi; Grace, Michael; Ukath, Jaysree; Lee, Vivienne S; McRobb, Lucinda S; Sedger, Lisa M; Stoodley, Marcus A

    2016-06-01

    OBJECT Stereotactic radiosurgery (SRS) is an established intervention for brain arteriovenous malformations (AVMs). The processes of AVM vessel occlusion after SRS are poorly understood. To improve SRS efficacy, it is important to understand the cellular response of blood vessels to radiation. The molecular changes on the surface of AVM endothelial cells after irradiation may also be used for vascular targeting. This study investigates radiation-induced externalization of phosphatidylserine (PS) on endothelial cells using live-cell imaging. METHODS An immortalized cell line generated from mouse brain endothelium, bEnd.3 cells, was cultured and irradiated at different radiation doses using a linear accelerator. PS externalization in the cells was subsequently visualized using polarity-sensitive indicator of viability and apoptosis (pSIVA)-IANBD, a polarity-sensitive probe. Live-cell imaging was used to monitor PS externalization in real time. The effects of radiation on the cell cycle of bEnd.3 cells were also examined by flow cytometry. RESULTS Ionizing radiation effects are dose dependent. Reduction in the cell proliferation rate was observed after exposure to 5 Gy radiation, whereas higher radiation doses (15 Gy and 25 Gy) totally inhibited proliferation. In comparison with cells treated with sham radiation, the irradiated cells showed distinct pseudopodial elongation with little or no spreading of the cell body. The percentages of pSIVA-positive cells were significantly higher (p = 0.04) 24 hours after treatment in the cultures that received 25- and 15-Gy doses of radiation. This effect was sustained until the end of the experiment (3 days). Radiation at 5 Gy did not induce significant PS externalization compared with the sham-radiation controls at any time points (p > 0.15). Flow cytometric analysis data indicate that irradiation induced growth arrest of bEnd.3 cells, with cells accumulating in the G2 phase of the cell cycle. CONCLUSIONS Ionizing radiation

  8. Brain Cancer Stem Cells: Current Status on Glioblastoma Multiforme

    International Nuclear Information System (INIS)

    Facchino, Sabrina; Abdouh, Mohamed; Bernier, Gilbert

    2011-01-01

    Glioblastoma multiforme (GBM), an aggressive brain tumor of astrocytic/neural stem cell origin, represents one of the most incurable cancers. GBM tumors are highly heterogeneous. However, most tumors contain a subpopulation of cells that display neural stem cell characteristics in vitro and that can generate a new brain tumor upon transplantation in mice. Hence, previously identified molecular pathways regulating neural stem cell biology were found to represent the cornerstone of GBM stem cell self-renewal mechanism. GBM tumors are also notorious for their resistance to radiation therapy. Notably, GBM “cancer stem cells” were also found to be responsible for this radioresistance. Herein, we will analyze the data supporting or not the cancer stem cell model in GBM, overview the current knowledge regarding GBM stem cell self-renewal and radioresistance molecular mechanisms, and discuss the potential therapeutic application of these findings

  9. A stable and reproducible human blood-brain barrier model derived from hematopoietic stem cells.

    Directory of Open Access Journals (Sweden)

    Romeo Cecchelli

    Full Text Available The human blood brain barrier (BBB is a selective barrier formed by human brain endothelial cells (hBECs, which is important to ensure adequate neuronal function and protect the central nervous system (CNS from disease. The development of human in vitro BBB models is thus of utmost importance for drug discovery programs related to CNS diseases. Here, we describe a method to generate a human BBB model using cord blood-derived hematopoietic stem cells. The cells were initially differentiated into ECs followed by the induction of BBB properties by co-culture with pericytes. The brain-like endothelial cells (BLECs express tight junctions and transporters typically observed in brain endothelium and maintain expression of most in vivo BBB properties for at least 20 days. The model is very reproducible since it can be generated from stem cells isolated from different donors and in different laboratories, and could be used to predict CNS distribution of compounds in human. Finally, we provide evidence that Wnt/β-catenin signaling pathway mediates in part the BBB inductive properties of pericytes.

  10. Affinity (tropism) of caprine arthritis encephalitis virus for brain cells ...

    African Journals Online (AJOL)

    The specific brain cell types infected by the (CAE) virus were determined using reverse-transcription polymerase chain reaction (RTPCR) and transmission electron microscopy (TEM techniques). TEM showed that in 85 – 90% cases, microglia were the cells specifically infected by the virus. Amplification of the genomic ...

  11. The brain microvascular endothelium supports T cell proliferation and has potential for alloantigen presentation.

    Directory of Open Access Journals (Sweden)

    Julie Wheway

    Full Text Available Endothelial cells (EC form the inner lining of blood vessels and are positioned between circulating lymphocytes and tissues. Hypotheses have formed that EC may act as antigen presenting cells based on the intimate interactions with T cells, which are seen in diseases like multiple sclerosis, cerebral malaria (CM and viral neuropathologies. Here, we investigated how human brain microvascular EC (HBEC interact with and support the proliferation of T cells. We found HBEC to express MHC II, CD40 and ICOSL, key molecules for antigen presentation and co-stimulation and to take up fluorescently labeled antigens via macropinocytosis. In co-cultures, we showed that HBEC support and promote the proliferation of CD4(+ and CD8(+ T cells, which both are key in CM pathogenesis, particularly following T cell receptor activation and co-stimulation. Our findings provide novel evidence that HBEC can trigger T cell activation, thereby providing a novel mechanism for neuroimmunological complications of infectious diseases.

  12. Human Brain Microvascular Endothelial Cells and Umbilical Vein Endothelial Cells Differentially Facilitate Leukocyte Recruitment and Utilize Chemokines for T Cell Migration

    Directory of Open Access Journals (Sweden)

    Shumei Man

    2008-01-01

    Full Text Available Endothelial cells that functionally express blood brain barrier (BBB properties are useful surrogates for studying leukocyte-endothelial cell interactions at the BBB. In this study, we compared two different endothelial cellular models: transfected human brain microvascular endothelial cells (THBMECs and human umbilical vein endothelial cells (HUVECs. With each grow under optimal conditions, confluent THBMEC cultures showed continuous occludin and ZO-1 immunoreactivity, while HUVEC cultures exhibited punctate ZO-1 expression at sites of cell-cell contact only. Confluent THBMEC cultures on 24-well collagen-coated transwell inserts had significantly higher transendothelial electrical resistance (TEER and lower solute permeability than HUVECs. Confluent THBMECs were more restrictive for mononuclear cell migration than HUVECs. Only THBMECs utilized abluminal CCL5 to facilitate T-lymphocyte migration in vitro although both THBMECs and HUVECs employed CCL3 to facilitate T cell migration. These data establish baseline conditions for using THBMECs to develop in vitro BBB models for studying leukocyte-endothelial interactions during neuroinflammation.

  13. Isolation and culture of larval cells from C. elegans.

    Directory of Open Access Journals (Sweden)

    Sihui Zhang

    Full Text Available Cell culture is an essential tool to study cell function. In C. elegans the ability to isolate and culture cells has been limited to embryonically derived cells. However, cells or blastomeres isolated from mixed stage embryos terminally differentiate within 24 hours of culture, thus precluding post-embryonic stage cell culture. We have developed an efficient and technically simple method for large-scale isolation and primary culture of larval-stage cells. We have optimized the treatment to maximize cell number and minimize cell death for each of the four larval stages. We obtained up to 7.8×10(4 cells per microliter of packed larvae, and up to 97% of adherent cells isolated by this method were viable for at least 16 hours. Cultured larval cells showed stage-specific increases in both cell size and multinuclearity and expressed lineage- and cell type-specific reporters. The majority (81% of larval cells isolated by our method were muscle cells that exhibited stage-specific phenotypes. L1 muscle cells developed 1 to 2 wide cytoplasmic processes, while L4 muscle cells developed 4 to 14 processes of various thicknesses. L4 muscle cells developed bands of myosin heavy chain A thick filaments at the cell center and spontaneously contracted ex vivo. Neurons constituted less than 10% of the isolated cells and the majority of neurons developed one or more long, microtubule-rich protrusions that terminated in actin-rich growth cones. In addition to cells such as muscle and neuron that are high abundance in vivo, we were also able to isolate M-lineage cells that constitute less than 0.2% of cells in vivo. Our novel method of cell isolation extends C. elegans cell culture to larval developmental stages, and allows use of the wealth of cell culture tools, such as cell sorting, electrophysiology, co-culture, and high-resolution imaging of subcellular dynamics, in investigation of post-embryonic development and physiology.

  14. Comparison of brain capillary endothelial cell-based and epithelial (MDCK-MDR1, Caco-2, and VB-Caco-2) cell-based surrogate blood-brain barrier penetration models.

    Science.gov (United States)

    Hellinger, Eva; Veszelka, Szilvia; Tóth, Andrea E; Walter, Fruzsina; Kittel, Agnes; Bakk, Mónika Laura; Tihanyi, Károly; Háda, Viktor; Nakagawa, Shinsuke; Duy, Thuy Dinh Ha; Niwa, Masami; Deli, Mária A; Vastag, Monika

    2012-10-01

    An accurate means of predicting blood-brain barrier (BBB) penetration and blood-brain partitioning of NCEs (new chemical entities) would fulfill a major need in pharmaceutical research. Currently, an industry-standard BBB drug penetration model is not available. Primary brain capillary endothelial cells, optionally co-cultured with astrocytes and/or pericytes, are the most valued models of BBB. For routine use, establishing and maintaining a co-culture system is too costly and labor intensive. Alternatively, non-cerebral cell lines such as MDCK-MDR1 are used, and most recently, the suitability of native and modified Caco-2 for predicting brain penetration has also come under investigation. This study provides comparative data on the morphology and functionality of the high integrity brain capillary endothelial BBB model (EPA: triple culture of brain capillary endothelial cells with pericytes and astrocytes) and the epithelial cell-based (native Caco-2, high P-glycoprotein expressing vinblastine-treated VB-Caco-2 and MDCK-MDR1) surrogate BBB models. Using a panel of 10 compounds VB-Caco-2 and MDCK-MDR1 cell lines show restrictive paracellular pathway and BBB-like selective passive permeability that makes them comparable to the rat brain BBB model, which gave correlation with the highest r(2) value with in vivo permeability data. In bidirectional assay, the VB-Caco-2 and the MDCK-MDR1 models identified more P-glycoprotein drug substrates than the rat brain BBB model. While the complexity and predictive value of the BBB model is the highest, for the screening of NCEs to determine whether they are efflux substrates or not, the VB-Caco-2 and the MDCK-MDR1 models may provide a simple and inexpensive tool. Copyright © 2012 Elsevier B.V. All rights reserved.

  15. Systems Biology for Organotypic Cell Cultures

    Energy Technology Data Exchange (ETDEWEB)

    Grego, Sonia [RTI International, Research Triangle Park, NC (United States); Dougherty, Edward R. [Texas A & M Univ., College Station, TX (United States); Alexander, Francis J. [Los Alamos National Lab. (LANL), Los Alamos, NM (United States); Auerbach, Scott S. [National Inst. of Environmental Health Sciences, Research Triangle Park, NC (United States); Berridge, Brian R. [GlaxoSmithKline, Research Triangle Park, NC (United States); Bittner, Michael L. [Translational Genomics Research Inst., Phoenix, AZ (United States); Casey, Warren [National Inst. of Environmental Health Sciences, Research Triangle Park, NC (United States); Cooley, Philip C. [RTI International, Research Triangle Park, NC (United States); Dash, Ajit [HemoShear Therapeutics, Charlottesville, VA (United States); Ferguson, Stephen S. [National Inst. of Environmental Health Sciences, Research Triangle Park, NC (United States); Fennell, Timothy R. [RTI International, Research Triangle Park, NC (United States); Hawkins, Brian T. [RTI International, Research Triangle Park, NC (United States); Hickey, Anthony J. [RTI International, Research Triangle Park, NC (United States); Kleensang, Andre [Johns Hopkins Univ., Baltimore, MD (United States). Center for Alternatives to Animal Testing; Liebman, Michael N. [IPQ Analytics, Kennett Square, PA (United States); Martin, Florian [Phillip Morris International, Neuchatel (Switzerland); Maull, Elizabeth A. [National Inst. of Environmental Health Sciences, Research Triangle Park, NC (United States); Paragas, Jason [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Qiao, Guilin [Defense Threat Reduction Agency, Ft. Belvoir, VA (United States); Ramaiahgari, Sreenivasa [National Inst. of Environmental Health Sciences, Research Triangle Park, NC (United States); Sumner, Susan J. [RTI International, Research Triangle Park, NC (United States); Yoon, Miyoung [The Hamner Inst. for Health Sciences, Research Triangle Park, NC (United States); ScitoVation, Research Triangle Park, NC (United States)

    2016-08-04

    Translating in vitro biological data into actionable information related to human health holds the potential to improve disease treatment and risk assessment of chemical exposures. While genomics has identified regulatory pathways at the cellular level, translation to the organism level requires a multiscale approach accounting for intra-cellular regulation, inter-cellular interaction, and tissue/organ-level effects. Tissue-level effects can now be probed in vitro thanks to recently developed systems of three-dimensional (3D), multicellular, “organotypic” cell cultures, which mimic functional responses of living tissue. However, there remains a knowledge gap regarding interactions across different biological scales, complicating accurate prediction of health outcomes from molecular/genomic data and tissue responses. Systems biology aims at mathematical modeling of complex, non-linear biological systems. We propose to apply a systems biology approach to achieve a computational representation of tissue-level physiological responses by integrating empirical data derived from organotypic culture systems with computational models of intracellular pathways to better predict human responses. Successful implementation of this integrated approach will provide a powerful tool for faster, more accurate and cost-effective screening of potential toxicants and therapeutics. On September 11, 2015, an interdisciplinary group of scientists, engineers, and clinicians gathered for a workshop in Research Triangle Park, North Carolina, to discuss this ambitious goal. Participants represented laboratory-based and computational modeling approaches to pharmacology and toxicology, as well as the pharmaceutical industry, government, non-profits, and academia. Discussions focused on identifying critical system perturbations to model, the computational tools required, and the experimental approaches best suited to generating key data. This consensus report summarizes the discussions held.

  16. Using Tissue Culture To Investigate Plant Cell Differentiation and Dedifferentiation.

    Science.gov (United States)

    Bozzone, Donna M.

    1997-01-01

    Describes an experimental project that uses plant tissue culture techniques to examine cell differentiation in the carrot. Allows students to gain experience in some important techniques and to explore fundamental questions about cell differentiation. (DDR)

  17. Synthesis of polymer materials for use as cell culture substrates

    Energy Technology Data Exchange (ETDEWEB)

    Lakard, Sophie [Laboratoire de Chimie des Materiaux et Interfaces, University of Franche-Comte, IUT, 30 Avenue de l' Observatoire, 25009 Besancon (France)], E-mail: sophie.lakard@univ-fcomte.fr; Morrand-Villeneuve, Nadege [Laboratoire de Neurosciences, University of Franche-Comte, Place Leclerc, 25030 Besancon (France); Lesniewska, Eric [Laboratoire de Physique de l' Universite de Bourgogne, University of Bourgogne, 9 Avenue Savary, 21078 Dijon (France); Lakard, Boris [Laboratoire de Chimie des Materiaux et Interfaces, University of Franche-Comte, 16 Route de Gray, 25030 Besancon (France); Michel, Germaine [Laboratoire de Neurosciences, University of Franche-Comte, Place Leclerc, 25030 Besancon (France); Herlem, Guillaume [Laboratoire de Chimie des Materiaux et Interfaces, University of Franche-Comte, 16 Route de Gray, 25030 Besancon (France); Gharbi, Tijani [Laboratoire d' Optique P.M. Duffieux, University of Franche-Comte, 16 Route de Gray, 25030 Besancon (France); Fahys, Bernard [Laboratoire de Chimie des Materiaux et Interfaces, University of Franche-Comte, 16 Route de Gray, 25030 Besancon (France)

    2007-12-20

    Up to today, several techniques have been used to maintain cells in culture for studying many aspects of cell biology and physiology. More often, cell culture is dependent on proper anchorage of cells to the growth surface. Thus, poly-L-lysine, fibronectin or laminin are the most commonly used substrates. In this study, electrosynthesized biocompatible polymer films are proposed as an alternative to these standard substrates. The electrosynthesized polymers tested were polyethylenimine, polypropylenimine and polypyrrole. Then, the adhesion, proliferation and morphology of rat neuronal cell lines were investigated on these polymer substrates in an attempt to develop new and efficient polymer materials for cell culture. During their growth on the polymers, the evolution of the cell morphology was monitored using both confocal microscopy and immunohistochemistry, leading to the conclusion of a normal development. An estimation of the adhesion and proliferation rates of rat neuronal cell cultures indicated that polyethylenimine and polypropylenimine were the best substrates for culturing olfactory neuronal cells. A method to favour the differentiation of the neuronal cells was also developed since the final aim of this work is to develop a biosensor for odour detection using differentiated neuronal cells as transducers. Consequently, a biosensor was microfabricated using silicon technology. This microsystem allowed us to culture the cells on a silicon wafer and to position the cells on certain parts of the silicon wafer.

  18. Development of a microfluidic perfusion 3D cell culture system

    Science.gov (United States)

    Park, D. H.; Jeon, H. J.; Kim, M. J.; Nguyen, X. D.; Morten, K.; Go, J. S.

    2018-04-01

    Recently, 3-dimensional in vitro cell cultures have gained much attention in biomedical sciences because of the closer relevance between in vitro cell cultures and in vivo environments. This paper presents a microfluidic perfusion 3D cell culture system with consistent control of long-term culture conditions to mimic an in vivo microenvironment. It consists of two sudden expansion reservoirs to trap incoming air bubbles, gradient generators to provide a linear concentration, and microchannel mixers. Specifically, the air bubbles disturb a flow in the microfluidic channel resulting in the instability of the perfusion cell culture conditions. For long-term stable operation, the sudden expansion reservoir is designed to trap air bubbles by using buoyancy before they enter the culture system. The performance of the developed microfluidic perfusion 3D cell culture system was examined experimentally and compared with analytical results. Finally, it was applied to test the cytotoxicity of cells infected with Ewing’s sarcoma. Cell death was observed for different concentrations of H2O2. For future work, the developed microfluidic perfusion 3D cell culture system can be used to examine the behavior of cells treated with various drugs and concentrations for high-throughput drug screening.

  19. Aeroponics for the culture of organisms, tissues and cells.

    Science.gov (United States)

    Weathers, P J; Zobel, R W

    1992-01-01

    Characteristics of aeroponics are discussed. Contrast is made, where appropriate, with hydroponics and aero-hydroponics as applies to research and commercial applications of nutrient mist technology. Topics include whole plants, plant tissue cultures, cell and microbial cultures, and animal tissue cultures with regard to operational considerations (moisture, temperature, minerals, gaseous atmosphere) and design of apparati.

  20. A method for culturing human hair follicle cells.

    Science.gov (United States)

    Weterings, P J; Vermorken, A J; Bloemendal, H

    1981-01-01

    For the first time a method for culturing human hair follicle cells is described. The bovine eye lens capsule, a basement membrane-like structure, is used as the substrate for the cultures. In a culture medium supplemented with hydrocortisone and insulin about 70% of the original follicles will form growing colonies of diploid keratinocytes.

  1. Initial contact of glioblastoma cells with existing normal brain endothelial cells strengthen the barrier function via fibroblast growth factor 2 secretion: a new in vitro blood-brain barrier model.

    Science.gov (United States)

    Toyoda, Keisuke; Tanaka, Kunihiko; Nakagawa, Shinsuke; Thuy, Dinh Ha Duy; Ujifuku, Kenta; Kamada, Kensaku; Hayashi, Kentaro; Matsuo, Takayuki; Nagata, Izumi; Niwa, Masami

    2013-05-01

    Glioblastoma multiforme (GBM) cells invade along the existing normal capillaries in brain. Normal capillary endothelial cells function as the blood-brain barrier (BBB) that limits permeability of chemicals into the brain. To investigate whether GBM cells modulate the BBB function of normal endothelial cells, we developed a new in vitro BBB model with primary cultures of rat brain endothelial cells (RBECs), pericytes, and astrocytes. Cells were plated on a membrane with 8 μm pores, either as a monolayer or as a BBB model with triple layer culture. The BBB model consisted of RBEC on the luminal side as a bottom, and pericytes and astrocytes on the abluminal side as a top of the chamber. Human GBM cell line, LN-18 cells, or lung cancer cell line, NCI-H1299 cells, placed on either the RBEC monolayer or the BBB model increased the transendothelial electrical resistance (TEER) values against the model, which peaked within 72 h after the tumor cell application. The TEER value gradually returned to baseline with LN-18 cells, whereas the value quickly dropped to the baseline in 24 h with NCI-H1299 cells. NCI-H1299 cells invaded into the RBEC layer through the membrane, but LN-18 cells did not. Fibroblast growth factor 2 (FGF-2) strengthens the endothelial cell BBB function by increased occludin and ZO-1 expression. In our model, LN-18 and NCI-H1299 cells secreted FGF-2, and a neutralization antibody to FGF-2 inhibited LN-18 cells enhanced BBB function. These results suggest that FGF-2 would be a novel therapeutic target for GBM in the perivascular invasive front.

  2. A cGMP-applicable expansion method for aggregates of human neural stem and progenitor cells derived from pluripotent stem cells or fetal brain tissue.

    Science.gov (United States)

    Shelley, Brandon C; Gowing, Geneviève; Svendsen, Clive N

    2014-06-15

    A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the stem cell community. Many current expansion methods are laborious and costly, and those involving complete dissociation may cause several stem and progenitor cell types to undergo differentiation or early senescence. To overcome these problems, we have developed an automated mechanical passaging method referred to as "chopping" that is simple and inexpensive. This technique avoids chemical or enzymatic dissociation into single cells and instead allows for the large-scale expansion of suspended, spheroid cultures that maintain constant cell/cell contact. The chopping method has primarily been used for fetal brain-derived neural progenitor cells or neurospheres, and has recently been published for use with neural stem cells derived from embryonic and induced pluripotent stem cells. The procedure involves seeding neurospheres onto a tissue culture Petri dish and subsequently passing a sharp, sterile blade through the cells effectively automating the tedious process of manually mechanically dissociating each sphere. Suspending cells in culture provides a favorable surface area-to-volume ratio; as over 500,000 cells can be grown within a single neurosphere of less than 0.5 mm in diameter. In one T175 flask, over 50 million cells can grow in suspension cultures compared to only 15 million in adherent cultures. Importantly, the chopping procedure has been used under current good manufacturing practice (cGMP), permitting mass quantity production of clinical-grade cell products.

  3. In vitro evidence for the brain glutamate efflux hypothesis: brain endothelial cells cocultured with astrocytes display a polarized brain-to-blood transport of glutamate.

    Science.gov (United States)

    Helms, Hans Christian; Madelung, Rasmus; Waagepetersen, Helle Sønderby; Nielsen, Carsten Uhd; Brodin, Birger

    2012-05-01

    The concentration of the excitotoxic amino acid, L-glutamate, in brain interstitial fluid is tightly regulated by uptake transporters and metabolism in astrocytes and neurons. The aim of this study was to investigate the possible role of the blood-brain barrier endothelium in brain L-glutamate homeostasis. Transendothelial transport- and accumulation studies of (3) H-L-glutamate, (3) H-L-aspartate, and (3) H-D-aspartate in an electrically tight bovine endothelial/rat astrocyte blood-brain barrier coculture model were performed. After 6 days in culture, the endothelium displayed transendothelial resistance values of 1014 ± 70 Ω cm(2) , and (14) C-D-mannitol permeability values of 0.88 ± 0.13 × 10(-6) cm s(-1) . Unidirectional flux studies showed that L-aspartate and L-glutamate, but not D-aspartate, displayed polarized transport in the brain-to-blood direction, however, all three amino acids accumulated in the cocultures when applied from the abluminal side. The transcellular transport kinetics were characterized with a K(m) of 69 ± 15 μM and a J(max) of 44 ± 3.1 pmol min(-1) cm(-2) for L-aspartate and a K(m) of 138 ± 49 μM and J(max) of 28 ± 3.1 pmol min(-1) cm(-2) for L-glutamate. The EAAT inhibitor, DL-threo-ß-Benzyloxyaspartate, inhibited transendothelial brain-to-blood fluxes of L-glutamate and L-aspartate. Expression of EAAT-1 (Slc1a3), -2 (Slc1a2), and -3 (Slc1a1) mRNA in the endothelial cells was confirmed by conventional PCR and localization of EAAT-1 and -3 in endothelial cells was shown with immunofluorescence. Overall, the findings suggest that the blood-brain barrier itself may participate in regulating brain L-glutamate concentrations. Copyright © 2012 Wiley Periodicals, Inc.

  4. Hypoxic stress up-regulates Kir2.1 expression and facilitates cell proliferation in brain capillary endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Yamamura, Hideto; Suzuki, Yoshiaki; Yamamura, Hisao [Department of Molecular & Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya (Japan); Asai, Kiyofumi [Department of Molecular Neurobiology, Graduate School of Medical Sciences, Nagoya City University, Nagoya (Japan); Imaizumi, Yuji, E-mail: yimaizum@phar.nagoya-cu.ac.jp [Department of Molecular & Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya (Japan)

    2016-08-05

    The blood-brain barrier (BBB) is mainly composed of brain capillary endothelial cells (BCECs), astrocytes and pericytes. Brain ischemia causes hypoxic encephalopathy and damages BBB. However, it remains still unclear how hypoxia affects BCECs. In the present study, t-BBEC117 cells, an immortalized bovine brain endothelial cell line, were cultured under hypoxic conditions at 4–5% oxygen for 72 h. This hypoxic stress caused hyperpolarization of resting membrane potential. Patch-clamp recordings revealed a marked increase in Ba{sup 2+}-sensitive inward rectifier K{sup +} current in t-BBEC117 cells after hypoxic culture. Western blot and real-time PCR analyses showed that Kir2.1 expression was significantly up-regulated at protein level but not at mRNA level after the hypoxic culture. Ca{sup 2+} imaging study revealed that the hypoxic stress enhanced store-operated Ca{sup 2+} (SOC) entry, which was significantly reduced in the presence of 100 μM Ba{sup 2+}. On the other hand, the expression of SOC channels such as Orai1, Orai2, and transient receptor potential channels was not affected by hypoxic stress. MTT assay showed that the hypoxic stress significantly enhanced t-BBEC117 cell proliferation, which was inhibited by approximately 60% in the presence of 100 μM Ba{sup 2+}. We first show here that moderate cellular stress by cultivation under hypoxic conditions hyperpolarizes membrane potential via the up-regulation of functional Kir2.1 expression and presumably enhances Ca{sup 2+} entry, resulting in the facilitation of BCEC proliferation. These findings suggest potential roles of Kir2.1 expression in functional changes of BCECs in BBB following ischemia. -- Highlights: •Hypoxic culture of brain endothelial cells (BEC) caused membrane hyperpolarization. •This hyperpolarization was due to the increased expression of Kir2.1 channels. •Hypoxia enhanced store-operated Ca{sup 2+} (SOC) entry via Kir2.1 up-regulation. •Expression levels of putative SOC

  5. Hypoxic stress up-regulates Kir2.1 expression and facilitates cell proliferation in brain capillary endothelial cells

    International Nuclear Information System (INIS)

    Yamamura, Hideto; Suzuki, Yoshiaki; Yamamura, Hisao; Asai, Kiyofumi; Imaizumi, Yuji

    2016-01-01

    The blood-brain barrier (BBB) is mainly composed of brain capillary endothelial cells (BCECs), astrocytes and pericytes. Brain ischemia causes hypoxic encephalopathy and damages BBB. However, it remains still unclear how hypoxia affects BCECs. In the present study, t-BBEC117 cells, an immortalized bovine brain endothelial cell line, were cultured under hypoxic conditions at 4–5% oxygen for 72 h. This hypoxic stress caused hyperpolarization of resting membrane potential. Patch-clamp recordings revealed a marked increase in Ba 2+ -sensitive inward rectifier K + current in t-BBEC117 cells after hypoxic culture. Western blot and real-time PCR analyses showed that Kir2.1 expression was significantly up-regulated at protein level but not at mRNA level after the hypoxic culture. Ca 2+ imaging study revealed that the hypoxic stress enhanced store-operated Ca 2+ (SOC) entry, which was significantly reduced in the presence of 100 μM Ba 2+ . On the other hand, the expression of SOC channels such as Orai1, Orai2, and transient receptor potential channels was not affected by hypoxic stress. MTT assay showed that the hypoxic stress significantly enhanced t-BBEC117 cell proliferation, which was inhibited by approximately 60% in the presence of 100 μM Ba 2+ . We first show here that moderate cellular stress by cultivation under hypoxic conditions hyperpolarizes membrane potential via the up-regulation of functional Kir2.1 expression and presumably enhances Ca 2+ entry, resulting in the facilitation of BCEC proliferation. These findings suggest potential roles of Kir2.1 expression in functional changes of BCECs in BBB following ischemia. -- Highlights: •Hypoxic culture of brain endothelial cells (BEC) caused membrane hyperpolarization. •This hyperpolarization was due to the increased expression of Kir2.1 channels. •Hypoxia enhanced store-operated Ca 2+ (SOC) entry via Kir2.1 up-regulation. •Expression levels of putative SOC channels were not affected by hypoxia.

  6. Mast cells in neuroinflammation and brain disorders

    NARCIS (Netherlands)

    Hendriksen, Erik; van Bergeijk, Doris; Oosting, Ronald S; Redegeld, Frank A

    2017-01-01

    It is well recognized that neuroinflammation is involved in the pathogenesis of various neurodegenerative diseases. Microglia and astrocytes are major pathogenic components within this process and known to respond to proinflammatory mediators released from immune cells such as mast cells. Mast cells

  7. Rabbit uterine epithelial cells: Co-culture with spermatozoa

    International Nuclear Information System (INIS)

    Boice, M.L.

    1988-01-01

    A primary culture of rabbit uterine epithelial cells was established and their effects on sperm function were examined in vitro. Epithelial cells were isolated from uteri of estrous rabbits and cultured on floating collagen gels in phenol red-free medium supplemented with 5% fetal bovine serum. Light microscopy and keratin staining showed that the epithelial cell population established in culture had morphological characteristics similar to that seen in the intact endometrium. Cells were cultured with 3 H-leucine and uptake of label by cells and its incorporation into cellular and secretory proteins determined. When compared to cells cultured for 24-48 h, incorporation of label into cellular protein was lower at 72-96 h, but secretion increased. Estradiol 17-β did not affect label uptake or incorporation, but did enhance proliferation of cells as judged by total DNA content of the cell population. Analysis of proteins in media by sodium dodecyl sulfate polyacrylamide gel electrophoresis and fluorography suggested that epithelial and stromal cells synthesis proteins that may be secretory in nature during 72-96 h culture. Twenty-nine to thirty-one h after initiation of epithelial cultures, 1-2 x 10 6 sperm were co-incubated with cells and sperm viability, motility, loss of acrosome and fertilizing ability determined

  8. Embodied Brains, Social Minds, Cultural Meaning: Integrating Neuroscientific and Educational Research on Social-Affective Development

    Science.gov (United States)

    Immordino-Yang, Mary Helen; Gotlieb, Rebecca

    2017-01-01

    Social-affective neuroscience is revealing that human brain development is inherently social--our very nature is organized by nurture. To explore the implications for human development and education, we present a series of interdisciplinary studies documenting individual and cultural variability in the neurobiological correlates of emotional…

  9. The Endo-Lysosomal System of Brain Endothelial Cells Is Influenced by Astrocytes In Vitro.

    Science.gov (United States)

    Toth, Andrea E; Siupka, Piotr; P Augustine, Thomas J; Venø, Susanne T; Thomsen, Louiza B; Moos, Torben; Lohi, Hannes T; Madsen, Peder; Lykke-Hartmann, Karin; Nielsen, Morten S

    2018-03-20

    Receptor- and adsorptive-mediated transport through brain endothelial cells (BEC) of the blood-brain barrier (BBB) involves a complex array of subcellular vesicular structures, the endo-lysosomal system. It consists of several types of vesicles, such as early, recycling, and late endosomes, retromer-positive structures, and lysosomes. Since this system is important for receptor-mediated transcytosis of drugs across brain capillaries, our aim was to characterise the endo-lysosomal system in BEC with emphasis on their interactions with astrocytes. We used primary porcine BEC in monoculture and in co-culture with primary rat astrocytes. The presence of astrocytes changed the intraendothelial vesicular network and significantly impacted vesicular number, morphology, and distribution. Additionally, gene set enrichment analysis revealed that 60 genes associated with vesicular trafficking showed altered expression in co-cultured BEC. Cytosolic proteins involved in subcellular trafficking were investigated to mark transport routes, such as RAB25 for transcytosis. Strikingly, the adaptor protein called AP1-μ1B, important for basolateral sorting in epithelial cells, was not expressed in BEC. Altogether, our data pin-point unique features of BEC trafficking network, essentially mapping the endo-lysosomal system of in vitro BBB models. Consequently, our findings constitute a valuable basis for planning the optimal route across the BBB when advancing drug delivery to the brain.

  10. Anatomy, technology, art, and culture: toward a realistic perspective of the brain.

    Science.gov (United States)

    Cavalcanti, Daniel D; Feindel, William; Goodrich, James T; Dagi, T Forcht; Prestigiacomo, Charles J; Preul, Mark C

    2009-09-01

    In the 15th century, brain illustration began to change from a schematic system that involved scant objective rendering of the brain, to accurate depictions based on anatomical dissections that demanded significant artistic talent. Notable examples of this innovation are the drawings of Leonardo da Vinci (1498-1504), Andreas Vesalius' association with the bottega of Titian to produce the drawings of Vesalius' De humani corporis fabrica (1543), and Christopher Wren's illustrations for Thomas Willis' Cerebri Anatome (1664). These works appeared during the Renaissance and Age of Enlightenment, when advances in brain imaging, or really brain rendering, reflected not only the abilities and dedications of the artists, but also the influences of important cultural and scientific factors. Anatomy and human dissection became popular social phenomena as well as scholarly pursuits, linked with the world of the fine arts. The working philosophy of these artists involved active participation in both anatomical study and illustration, and the belief that their discoveries of the natural world could best be communicated by rendering them in objective form (that is, with realistic perspective). From their studies emerged the beginning of contemporary brain imaging. In this article, the authors examine how the brain began to be imaged in realism within a cultural and scientific milieu that witnessed the emergence of anatomical dissection, the geometry of linear perspective, and the closer confluence of art and science.

  11. Biocompatibility of silicon-based arrays of electrodes coupled to organotypic hippocampal brain slice cultures

    DEFF Research Database (Denmark)

    Kristensen, Bjarne Winther; Noraberg, J; Thiébaud, P

    2001-01-01

    In this study we examined the passive biocompatibility of a three-dimensional microelectrode array (MEA), designed to be coupled to organotypic brain slice cultures for multisite recording of electrophysiological signals. Hippocampal (and corticostriatal) brain slices from 1-week-old (and newborn......) rats were grown for 4-8 weeks on the perforated silicon chips with silicon nitride surfaces and 40 microm sized holes and compared with corresponding tissue slices grown on conventional semiporous membranes. In terms of preservation of the basic cellular and connective organization, as visualized......-methyl-D-aspartate (NMDA) and the neurotoxin trimethyltin (TMT), as demonstrated by the cellular uptake of propidium iodide (PI), which was used as a reproducible and quantifiable marker for neuronal degeneration. We conclude that organotypic brain slice cultures can grow on silicon-based three-dimensional microelectrode...

  12. Microfluidic culture chamber for the long-term perfusion and precise chemical stimulation of organotypic brain tissue slices

    DEFF Research Database (Denmark)

    Caicedo, H. H.; Vignes, M.; Brugg, B.

    2010-01-01

    We have developed a microfluidic perfusion-based culture system to study long-term in-vitro responses of organo-typic brain slices exposed to localized neurochemical stimulation. Using this microperfusion chamber we show that hip-pocampal organotypic brain slices cultures grown on nitrocellulose ...

  13. Growth and Plating of Cell Suspension Cultures of Datura Innoxia

    DEFF Research Database (Denmark)

    Engvild, Kjeld Christensen

    1974-01-01

    malate) or on NO3−-N alone. Dry weight yield was proportional to the amount of nitrate-N added (47 mg/mg N). Filtered suspension cultures containing single cells (plating cultures) could be grown in agar in petri dishes when NAA or 2,4-D were used as growth substances. Cells grew at densities above 500...

  14. Rapid method for culturing embryonic neuron-glial cell cocultures

    DEFF Research Database (Denmark)

    Svenningsen, Åsa Fex; Shan, Wei-Song; Colman, David R

    2003-01-01

    A streamlined, simple technique for primary cell culture from E17 rat tissue is presented. In an attempt to standardize culturing methods for all neuronal cell types in the embryo, we evaluated a commercial medium without serum and used similar times for trypsinization and tested different surfaces...

  15. Induction of interdigitating cell processes in podocyte culture.

    Science.gov (United States)

    Yaoita, Eishin; Yoshida, Yutaka; Nameta, Masaaki; Takimoto, Hiroki; Fujinaka, Hidehiko

    2018-02-01

    Highly organized cell processes characterize glomerular podocytes in vivo. However, podocytes in culture have a simple morphology lacking cell processes, especially upon reaching confluence. Here, we aimed to establish culture conditions under which cultured podocytes extend cell processes at confluence. Among various culture conditions that could possibly cause phenotypic changes in podocytes, we examined the effects of heparin, all-trans retinoic acid, fetal bovine serum, and extracellular matrices on the morphology of podocytes in rat primary culture. Consequently, long arborized cell processes were observed to radiate extensively from the cell body only when cells were cultured in the presence of heparin and all-trans retinoic acid on laminin-coated dishes with decreasing concentrations of fetal bovine serum. Primary processes branching repeatedly into terminal processes and cell process insertion under adjacent cell bodies were evident by electron microscopy-based analysis. Immunostaining for podocin showed conspicuous elongations of intercellular junctions. Under these conditions, the expression levels of podocyte-specific proteins and genes were markedly upregulated. Thus, we succeeded in establishing culture conditions in which the cultured podocytes exhibit phenotypes similar to those under in vivo conditions. Copyright © 2017 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

  16. Transplanted Neural Progenitor Cells from Distinct Sources Migrate Differentially in an Organotypic Model of Brain Injury

    Directory of Open Access Journals (Sweden)

    Kapinga P. Ngalula

    2015-10-01

    Full Text Available Brain injury is a major cause of long-term disability. The possibility exists for exogenously derived neural progenitor cells to repair damage resulting from brain injury, although more information is needed to successfully implement this promising therapy. To test the ability of neural progenitor cells (NPCs obtained from rats to repair damaged neocortex, we transplanted neural progenitor cell suspensions into normal and injured slice cultures of the neocortex acquired from rats on postnatal day 0–3. Donor cells from E16 embryos were obtained from either the neocortex, including the ventricular zone (VZ for excitatory cells, ganglionic eminence (GE for inhibitory cells or a mixed population of the two. Cells were injected into the ventricular/subventricular zone (VZ/SVZ or directly into the wounded region. Transplanted cells migrated throughout the cortical plate with GE and mixed population donor cells predominately targeting the upper cortical layers, while neocortically derived NPCs from the VZ/SVZ migrated less extensively. In the injured neocortex, transplanted cells moved predominantly into the wounded area. NPCs derived from the GE tended to be immunoreactive for GABAergic markers while those derived from the neocortex were more strongly immunoreactive for other neuronal markers such as MAP2, TUJ1, or Milli-Mark. Cells transplanted in vitro acquired the electrophysiological characteristics of neurons, including action potential generation and reception of spontaneous synaptic activity. This suggests that transplanted cells differentiate into neurons capable of functionally integrating with the host tissue. Together, our data suggest that transplantation of neural progenitor cells holds great potential as an emerging therapeutic intervention for restoring function lost to brain damage.

  17. Viable Cell Culture Banking for Biodiversity Characterization and Conservation.

    Science.gov (United States)

    Ryder, Oliver A; Onuma, Manabu

    2018-02-15

    Because living cells can be saved for indefinite periods, unprecedented opportunities for characterizing, cataloging, and conserving biological diversity have emerged as advanced cellular and genetic technologies portend new options for preventing species extinction. Crucial to realizing the potential impacts of stem cells and assisted reproductive technologies on biodiversity conservation is the cryobanking of viable cell cultures from diverse species, especially those identified as vulnerable to extinction in the near future. The advent of in vitro cell culture and cryobanking is reviewed here in the context of biodiversity collections of viable cell cultures that represent the progress and limitations of current efforts. The prospects for incorporating collections of frozen viable cell cultures into efforts to characterize the genetic changes that have produced the diversity of species on Earth and contribute to new initiatives in conservation argue strongly for a global network of facilities for establishing and cryobanking collections of viable cells.

  18. Horizontally rotated cell culture system with a coaxial tubular oxygenator

    Science.gov (United States)

    Wolf, David A. (Inventor); Schwarz, Ray P. (Inventor); Trinh, Tinh T. (Inventor)

    1991-01-01

    The present invention relates to a horizontally rotating bioreactor useful for carrying out cell and tissue culture. For processing of mammalian cells, the system is sterilized and fresh fluid medium, microcarrier beads, and cells are admitted to completely fill the cell culture vessel. An oxygen containing gas is admitted to the interior of the permeable membrane which prevents air bubbles from being introduced into the medium. The cylinder is rotated at a low speed within an incubator so that the circular motion of the fluid medium uniformly suspends the microbeads throughout the cylinder during the cell growth period. The unique design of this cell and tissue culture device was initially driven by two requirements imposed by its intended use for feasibility studies for three dimensional culture of living cells and tissues in space by JSC. They were compatible with microgravity and simulation of microgravity in one G. The vessels are designed to approximate the extremely quiescent low shear environment obtainable in space.

  19. The release of iron by Sertoli cells in culture

    NARCIS (Netherlands)

    Wauben-Penris, P. J.; Veldscholte, J.; van der Ende, A.; van der Donk, H. A.

    1988-01-01

    In seminiferous tubules, iron transport from the blood to the abluminal germinal cells must occur through the Sertoli cell cytoplasm. We investigated the release of previously accumulated iron by cultured Sertoli cells. We found that Sertoli cells contain easily releasable and less easily releasable

  20. PECULIARITIES OF SECONDARY METABOLITES BIOSYNTHESIS IN PLANT CELL CULTURES

    Directory of Open Access Journals (Sweden)

    A.M. NOSOV

    2014-06-01

    Full Text Available metabolites formation in plant cell cultures of Panax spp., (ginsenosides; Dioscorea deltoidea (steroid glycosides; Ajuga reptans, Serratula coronata, Rhaponticum carthamoides (ecdisteroids; Polyscias spp., (triterpene glycosides, Taxus spp. (taxoids, Stevia rebaudiana (diterpene steviol-glycosides, Stephania glabra (alkaloids. They are some regular trends of secondary metabolites synthesis in the plant cell culture:It can be noted the stable synthesis of the compound promoting cell proliferation. Indeed, cell cultures of Dioscorea deltoidea were demonstrated to accumulate only furostanol glycosides, which promoted cell division. Furostanol glycoside content of Dioscorea strain DM-0.5 was up to 6 - 12% by dry biomass.Panax ginseng and P. japonicus plant cell cultures synthesize as minimum seven triterpene glycosides (ginsenosides, the productivity of these compounds was up to 6.0 - 8.0% on dry biomass.By contrast, the detectable synthesis of diterpene steviol-glycosides in cultivated cells of Stevia rebaudiana initiated in the mixotrophic cultures during chloroplast formation only.Despite these differences, or mainly due to them, plant cell cultures have become an attractive source of phytochemicals in alternative to collecting wild plants. It provides a guideline to bioreactor-based production of isoprenoids using undifferentiated plant cell cultures

  1. Bidirectional apical-basal traffic of the cation-independent mannose-6-phosphate receptor in brain endothelial cells

    DEFF Research Database (Denmark)

    Siupka, Piotr; Hersom, Maria N. S.; Lykke-Hartmann, Karin

    2017-01-01

    in the process. Here, we elucidate the endosomal trafficking of the retrograde transported cation-independent mannose-6-phosphate receptor (MPR300) in primary cultures of brain endothelial cells (BECs) of porcine and bovine origin. Receptor expression and localisation of MPR300 in the endo-lysosomal system...

  2. Cell Delivery System for Traumatic Brain Injury

    Science.gov (United States)

    2008-03-21

    Injury Using Novel Matrices and Human Bone Marrow Stem Cells.” 4th Annual Los Angeles Tissue Engineering Meeting, UCLA Dec. 2006. (c) Presentations...Task 1). Task 1: Differentiate Adult Stem Cells into Neurons. Each of three different adult stem cell types (ADSCs, MSCs and amniotic -derived...gel properties. Evaluate gel material properties such as liquid to gel transition temperature, fiber and pore sizes, mechanical strength, resistance

  3. Patient-derived stem cells: pathways to drug discovery for brain diseases

    Directory of Open Access Journals (Sweden)

    Alan eMackay-Sim

    2013-03-01

    Full Text Available The concept of drug discovery through stem cell biology is based on technological developments whose genesis is now coincident. The first is automated cell microscopy with concurrent advances in image acquisition and analysis, known as high content screening (HCS. The second is patient-derived stem cells for modelling the cell biology of brain diseases. HCS has developed from the requirements of the pharmaceutical industry for high throughput assays to screen thousands of chemical compounds in the search for new drugs. HCS combines new fluorescent probes with automated microscopy and computational power to quantify the effects of compounds on cell functions. Stem cell biology has advanced greatly since the discovery of genetic reprogramming of somatic cells into induced pluripotent stem cells (iPSCs. There is now a rush of papers describing their generation from patients with various diseases of the nervous system. Although the majority of these have been genetic diseases, iPSCs have been generated from patients with complex diseases (schizophrenia and sporadic Parkinson’s disease. Some genetic diseases are also modelled in embryonic stem cells generated from blastocysts rejected during in vitro fertilisation. Neural stem cells have been isolated from post-mortem brain of Alzheimer’s patients and neural stem cells generated from biopsies of the olfactory organ of patients is another approach. These olfactory neurosphere-derived cells demonstrate robust disease-specific phenotypes in patients with schizophrenia and Parkinson’s disease. High content screening is already in use to find small molecules for the generation and differentiation of embryonic stem cells and induced pluripotent stem cells. The challenges for using stem cells for drug discovery are to develop robust stem cell culture methods that meet the rigorous requirements for repeatable, consistent quantities of defined cell types at the industrial scale necessary for high

  4. Radiosensitivity of normal human epidermal cells in culture

    International Nuclear Information System (INIS)

    Dover, R.; Potten, C.S.

    1983-01-01

    Using an in vitro culture system the authors have derived #betta#-radiation survival curves over a dose range 0-8 Gy for the clonogenic cells of normal human epidermis. The culture system used allows the epidermal cells to stratify and form a multi-layered sheet of keratinizing cells. The cultures appear to be a very good model for epidermis in vivo. The survival curves show a population which is apparently more sensitive than murine epidermis in vivo. It remains unclear whether this is an intrinsic difference between the species or is a consequence of the in vitro cultivation of the human cells. (author)

  5. Spatial organization and correlations of cell nuclei in brain tumors.

    Directory of Open Access Journals (Sweden)

    Yang Jiao

    Full Text Available Accepting the hypothesis that cancers are self-organizing, opportunistic systems, it is crucial to understand the collective behavior of cancer cells in their tumorous heterogeneous environment. In the present paper, we ask the following basic question: Is this self-organization of tumor evolution reflected in the manner in which malignant cells are spatially distributed in their heterogeneous environment? We employ a variety of nontrivial statistical microstructural descriptors that arise in the theory of heterogeneous media to characterize the spatial distributions of the nuclei of both benign brain white matter cells and brain glioma cells as obtained from histological images. These descriptors, which include the pair correlation function, structure factor and various nearest neighbor functions, quantify how pairs of cell nuclei are correlated in space in various ways. We map the centroids of the cell nuclei into point distributions to show that while commonly used local spatial statistics (e.g., cell areas and number of neighboring cells cannot clearly distinguish spatial correlations in distributions of normal and abnormal cell nuclei, their salient structural features are captured very well by the aforementioned microstructural descriptors. We show that the tumorous cells pack more densely than normal cells and exhibit stronger effective repulsions between any pair of cells. Moreover, we demonstrate that brain gliomas are organized in a collective way rather than randomly on intermediate and large length scales. The existence of nontrivial spatial correlations between the abnormal cells strongly supports the view that cancer is not an unorganized collection of malignant cells but rather a complex emergent integrated system.

  6. Spatial organization and correlations of cell nuclei in brain tumors.

    Science.gov (United States)

    Jiao, Yang; Berman, Hal; Kiehl, Tim-Rasmus; Torquato, Salvatore

    2011-01-01

    Accepting the hypothesis that cancers are self-organizing, opportunistic systems, it is crucial to understand the collective behavior of cancer cells in their tumorous heterogeneous environment. In the present paper, we ask the following basic question: Is this self-organization of tumor evolution reflected in the manner in which malignant cells are spatially distributed in their heterogeneous environment? We employ a variety of nontrivial statistical microstructural descriptors that arise in the theory of heterogeneous media to characterize the spatial distributions of the nuclei of both benign brain white matter cells and brain glioma cells as obtained from histological images. These descriptors, which include the pair correlation function, structure factor and various nearest neighbor functions, quantify how pairs of cell nuclei are correlated in space in various ways. We map the centroids of the cell nuclei into point distributions to show that while commonly used local spatial statistics (e.g., cell areas and number of neighboring cells) cannot clearly distinguish spatial correlations in distributions of normal and abnormal cell nuclei, their salient structural features are captured very well by the aforementioned microstructural descriptors. We show that the tumorous cells pack more densely than normal cells and exhibit stronger effective repulsions between any pair of cells. Moreover, we demonstrate that brain gliomas are organized in a collective way rather than randomly on intermediate and large length scales. The existence of nontrivial spatial correlations between the abnormal cells strongly supports the view that cancer is not an unorganized collection of malignant cells but rather a complex emergent integrated system.

  7. Tryptophan oxidation catabolite, N-formylkynurenine, in photo degraded cell culture medium results in reduced cell culture performance.

    Science.gov (United States)

    McElearney, Kyle; Ali, Amr; Gilbert, Alan; Kshirsagar, Rashmi; Zang, Li

    2016-01-01

    Chemically defined media have been widely used in the biopharmaceutical industry to enhance cell culture productivities and ensure process robustness. These media, which are quite complex, often contain a mixture of many components such as vitamins, amino acids, metals and other chemicals. Some of these components are known to be sensitive to various stress factors including photodegradation. Previous work has shown that small changes in impurity concentrations induced by these potential stresses can have a large impact on the cell culture process including growth and product quality attributes. Furthermore, it has been shown to be difficult to detect these modifications analytically due to the complexity of the cell culture media and the trace level of the degradant products. Here, we describe work performed to identify the specific chemical(s) in photodegraded medium that affect cell culture performance. First, we developed a model system capable of detecting changes in cell culture performance. Second, we used these data and applied an LC-MS analytical technique to characterize the cell culture media and identify degradant products which affect cell culture performance. Riboflavin limitation and N-formylkynurenine (NFK), a tryptophan oxidation catabolite, were identified as chemicals which results in a reduction in cell culture performance. © 2015 American Institute of Chemical Engineers.

  8. A population of human brain cells expressing phenotypic markers of more than one lineage can be induced in vitro to differentiate into mesenchymal cells

    International Nuclear Information System (INIS)

    Rieske, Piotr; Augelli, Brian J.; Stawski, Robert; Gaughan, John; Azizi, S. Ausim; Krynska, Barbara

    2009-01-01

    Proliferating astrocytic cells from germinal, as well as mature areas of brain parenchyma, have the characteristics of neural stem/progenitor cells and are capable of generating both neurons and glia. We previously reported that primary fetal human brain cells, designated as Normal Human Astrocytes (NHA), expressed, in addition to GFAP, Vimentin and Nestin, low levels of βIII-Tubulin, an early neuronal marker, and differentiated into neurons and astrocytes in vitro. Here, we showed that primary NHA cells co-express low levels of mesenchymal markers Fibronectin and Collagen-1 in culture. These cells transitioned into mesenchymal-like cells when cultured in adherent conditions in serum containing media. The mesenchymal-like derivatives of these cells were characterized based on their morphological changes, high expression of Vimentin and extracellular matrix (ECM) proteins, Collagen-1 and Fibronectin, and decline of neural markers. When incubated in osteogenic and adipogenic induction media, the mesenchymal-like cells differentiated into osteoblasts and adipocytes. Furthermore, NHA cells express markers of neural crest cells, SOX-10 and p75. These data support the idea of ectoderm-derived mesenchymal lineages. These findings suggest that a population of primitive fetal brain cells with neural/neural crest/mesenchymal phenotype, resembles the remarkable phenotypic plasticity of neural crest cells, and differentiates into adipocytes and osteocytes under the influence of environmental factors

  9. A kinetic model for flavonoid production in tea cell culture.

    Science.gov (United States)

    Shibasaki-Kitakawa, Naomi; Iizuka, Yasuhiro; Takahashi, Atsushi; Yonemoto, Toshikuni

    2017-02-01

    As one of the strategies for efficient production of a metabolite from cell cultures, a kinetic model is very useful tool to predict productivity under various culture conditions. In this study, we propose a kinetic model for flavonoid production in tea cell culture based on the cell life cycle and expression of PAL, the gene encoding phenylalanine ammonia-lyase (PAL)-the key enzyme in flavonoid biosynthesis. The flavonoid production rate was considered to be related to the amount of active PAL. Synthesis of PAL was modelled based on a general gene expression/translation mechanism, including the transcription of DNA encoding PAL into mRNA and the translation of PAL mRNA into the PAL protein. The transcription of DNA was assumed to be promoted at high light intensity and suppressed by a feedback regulatory mechanism at high flavonoid concentrations. In the model, mRNA and PAL were considered to self-decompose and to be lost by cell rupture. The model constants were estimated by fitting the experimental results obtained from tea cell cultures under various light intensities. The model accurately described the kinetic behaviors of dry and fresh cell concentrations, glucose concentration, cell viability, PAL specific activity, and flavonoid content under a wide range of light intensities. The model simulated flavonoid productivity per medium under various culture conditions. Therefore, this model will be useful to predict optimum culture conditions for maximum flavonoid productivity in cultured tea cells.

  10. [Application of cell co-culture techniques in medical studies].

    Science.gov (United States)

    Luo, Yun; Sun, Gui-Bo; Qin, Meng; Yao, Fan; Sun, Xiao-Bo

    2012-11-01

    As the cell co-culture techniques can better imitate an in vivo environment, it is helpful in observing the interactions among cells and between cells and the culture environment, exploring the effect mechanisms of drugs and their possible targets and filling the gaps between the mono-layer cell culture and the whole animal experiments. In recently years, they has attracted much more attention from the medical sector, and thus becoming one of research hotspots in drug research and development and bio-pharmaceutical fields. The cell co-culture techniques, including direct and indirect methods, are mainly used for studying pathological basis, new-type treatment methods and drug activity screening. Existing cell co-culture techniques are used for more pharmacological studies on single drug and less studies on interaction of combined drugs, such as collaborative compatibility and attenuation and synergistic effect among traditional Chinese medicines (TCMs). In line with the action characteristics of multi-component and multi-target, the cell co-culture techniques provide certain reference value for future studies on the effect and mechanism of combined TCMs on organisms as well as new methods for studies on TCMs and their compounds. This essay summarizes cell co-culture methods and their application and look into the future of their application in studies on TCMs and compounds.

  11. 21 CFR 876.5885 - Tissue culture media for human ex vivo tissue and cell culture processing applications.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Tissue culture media for human ex vivo tissue and... DEVICES Therapeutic Devices § 876.5885 Tissue culture media for human ex vivo tissue and cell culture processing applications. (a) Identification. Tissue culture media for human ex vivo tissue and cell culture...

  12. Radiosensitivity of primary cultured fish cells with different ploidy

    International Nuclear Information System (INIS)

    Mitani, Hiroshi; Egami, Nobuo; Kobayashi, Hiromu.

    1986-01-01

    The radiosensitivity of primary cultured goldfish cells (Carassius auratus) was investigated by colony formation assay. The radiosensitivity of cells from two varieties of goldfish, which show different sensitivity to lethal effect of ionizing radiation in vivo, was almost identical. Primary cultured cells from diploid, triploid and tetraploid fish retained their DNA content as measured by microfluorometry, and the nuclear size increases as ploidy increases. However, radiosensitivity was not related to ploidy. (author)

  13. Treatment of Mycoplasma Contamination in Cell Cultures with Plasmocin

    OpenAIRE

    Uphoff, Cord C.; Denkmann, Sabine-A.; Drexler, Hans G.

    2012-01-01

    A high percentage of cell lines are chronically infected with various mycoplasma species. The addition of antibiotics that are particularly effective against these contaminants to the culture medium during a limited period of time is a simple, inexpensive, and very practical approach for decontaminating cell cultures. Here, we examined the effectiveness of the new antimycoplasma compound Plasmocin that has been employed routinely to cleanse chronically infected cell lines. In a first round of...

  14. Multizone Paper Platform for 3D Cell Cultures

    Science.gov (United States)

    Derda, Ratmir; Hong, Estrella; Mwangi, Martin; Mammoto, Akiko; Ingber, Donald E.; Whitesides, George M.

    2011-01-01

    In vitro 3D culture is an important model for tissues in vivo. Cells in different locations of 3D tissues are physiologically different, because they are exposed to different concentrations of oxygen, nutrients, and signaling molecules, and to other environmental factors (temperature, mechanical stress, etc). The majority of high-throughput assays based on 3D cultures, however, can only detect the average behavior of cells in the whole 3D construct. Isolation of cells from specific regions of 3D cultures is possible, but relies on low-throughput techniques such as tissue sectioning and micromanipulation. Based on a procedure reported previously (“cells-in-gels-in-paper” or CiGiP), this paper describes a simple method for culture of arrays of thin planar sections of tissues, either alone or stacked to create more complex 3D tissue structures. This procedure starts with sheets of paper patterned with hydrophobic regions that form 96 hydrophilic zones. Serial spotting of cells suspended in extracellular matrix (ECM) gel onto the patterned paper creates an array of 200 micron-thick slabs of ECM gel (supported mechanically by cellulose fibers) containing cells. Stacking the sheets with zones aligned on top of one another assembles 96 3D multilayer constructs. De-stacking the layers of the 3D culture, by peeling apart the sheets of paper, “sections” all 96 cultures at once. It is, thus, simple to isolate 200-micron-thick cell-containing slabs from each 3D culture in the 96-zone array. Because the 3D cultures are assembled from multiple layers, the number of cells plated initially in each layer determines the spatial distribution of cells in the stacked 3D cultures. This capability made it possible to compare the growth of 3D tumor models of different spatial composition, and to examine the migration of cells in these structures. PMID:21573103

  15. Perivascular Mesenchymal Stem Cells From the Adult Human Brain Harbor No Instrinsic Neuroectodermal but High Mesodermal Differentiation Potential.

    Science.gov (United States)

    Lojewski, Xenia; Srimasorn, Sumitra; Rauh, Juliane; Francke, Silvan; Wobus, Manja; Taylor, Verdon; Araúzo-Bravo, Marcos J; Hallmeyer-Elgner, Susanne; Kirsch, Matthias; Schwarz, Sigrid; Schwarz, Johannes; Storch, Alexander; Hermann, Andreas

    2015-10-01

    Brain perivascular cells have recently been identified as a novel mesodermal cell type in the human brain. These cells reside in the perivascular niche and were shown to have mesodermal and, to a lesser extent, tissue-specific differentiation potential. Mesenchymal stem cells (MSCs) are widely proposed for use in cell therapy in many neurological disorders; therefore, it is of importance to better understand the "intrinsic" MSC population of the human brain. We systematically characterized adult human brain-derived pericytes during in vitro expansion and differentiation and compared these cells with fetal and adult human brain-derived neural stem cells (NSCs) and adult human bone marrow-derived MSCs. We found that adult human brain pericytes, which can be isolated from the hippocampus and from subcortical white matter, are-in contrast to adult human NSCs-easily expandable in monolayer cultures and show many similarities to human bone marrow-derived MSCs both regarding both surface marker expression and after whole transcriptome profile. Human brain pericytes showed a negligible propensity for neuroectodermal differentiation under various differentiation conditions but efficiently generated mesodermal progeny. Consequently, human brain pericytes resemble bone marrow-derived MSCs and might be very interesting for possible autologous and endogenous stem cell-based treatment strategies and cell therapeutic approaches for treating neurological diseases. Perivascular mesenchymal stem cells (MSCs) recently gained significant interest because of their appearance in many tissues including the human brain. MSCs were often reported as being beneficial after transplantation in the central nervous system in different neurological diseases; therefore, adult brain perivascular cells derived from human neural tissue were systematically characterized concerning neural stem cell and MSC marker expression, transcriptomics, and mesodermal and inherent neuroectodermal differentiation

  16. Feeding Frequency Affects Cultured Rat Pituitary Cells in Low Gravity

    Science.gov (United States)

    Hymer, W. C.; Grindeland, R. E.; Salada, T.; Cenci, R.; Krishnan, K.; Mukai, C.; Nagaoka, S.

    1996-01-01

    In this report, we describe the results of a rat pituitary cell culture experiment done on STS-65 in which the effect of cell feeding on the release of the six anterior pituitary hormones was studied. We found complex microgravity related interactions between the frequency of cell feeding and the quantity and quality (i.e. biological activity) of some of the six hormones released in flight. Analyses of growth hormone (GH) released from cells into culture media on different mission days using gel filtration and ion exchange chromatography yielded qualitatively similar results between ground and flight samples. Lack of cell feeding resulted in extensive cell clumping in flight (but not ground) cultures. Vigorous fibroblast growth occurred in both ground and flight cultures fed 4 times. These results are interpreted within the context of autocrine and or paracrine feedback interactions. Finally the payload specialist successfully prepared a fresh trypsin solution in microgravity, detached the cells from their surface and reinserted them back into the culture chamber. These cells reattached and continued to release hormone in microgravity. In summary, this experiment shows that pituitary cells are microgravity sensitive and that coupled operations routinely associated with laboratory cel1 culture can also be accomplished in low gravity.

  17. Radiation adaptive response for the growth of cultured glial cells

    International Nuclear Information System (INIS)

    Suzuki, S.; Miura, Y.; Kano, M.; Toda, T.; Urano, S.

    2003-01-01

    Full text: To examine the molecular mechanism of radiation adaptive response (RAR) for the growth of cultured glial cells and to investigate the influence of aging on the response, glial cells were cultured from young and aged rats (1 month and 24 months old). RAR for the growth of glial cells conditioned with a low dose of X-rays and subsequently exposed to a high dose of X-rays was examined for cell number and BrdU incorporation. Involvement of the subcellular signaling pathway factors in RAR was investigated using their inhibitors, activators and mutated glial cells. RAR was observed in cells cultured from young rats, but was not in cells from aged rats. The inhibitors of protein kinase C (PKC) and DNA-dependent protein kinase (DNA-PK) or phosphatidylinositol 3-kinase (PI3K) suppressed RAR. The activators of PKC instead of low dose irradiation also caused RAR. Moreover, glial cells cultured from severe combined immunodeficiency (scid) mice (CB-17 scid) and ataxia-telangiectasia (AT) cells from AT patients showed no RAR. These results indicated that PKC, ATM, DNAPK and/or PI3K were involved in RAR for growth and BrdU incorporation of cultured glial cells and RAR decreased with aging. Proteomics data of glial cells exposed to severe stress of H 2 O 2 or X-rays also will be presented in the conference since little or no difference has not been observed with slight stress yet

  18. Growth hormone (GH), brain development and neural stem cells.

    Science.gov (United States)

    Waters, M J; Blackmore, D G

    2011-12-01

    A range of observations support a role for GH in development and function of the brain. These include altered brain structure in GH receptor null mice, and impaired cognition in GH deficient rodents and in a subgroup of GH receptor defective patients (Laron dwarfs). GH has been shown to alter neurogenesis, myelin synthesis and dendritic branching, and both the GH receptor and GH itself are expressed widely in the brain. We have found a population of neural stem cells which are activated by GH infusion, and which give rise to neurons in mice. These stem cells are activated by voluntary exercise in a GH-dependent manner. Given the findings that local synthesis of GH occurs in the hippocampus in response to a memory task, and that GH replacement improves memory and cognition in rodents and humans, these new observations warrant a reappraisal of the clinical importance of GH replacement in GH deficient states.

  19. Solid Lipid Curcumin Particles Induce More DNA Fragmentation and Cell Death in Cultured Human Glioblastoma Cells than Does Natural Curcumin

    Directory of Open Access Journals (Sweden)

    Panchanan Maiti

    2017-01-01

    Full Text Available Despite recent advancements in cancer therapies, glioblastoma multiforme (GBM remains largely incurable. Curcumin (Cur, a natural polyphenol, has potent anticancer effects against several malignancies, including metastatic brain tumors. However, its limited bioavailability reduces its efficiency for treating GBM. Recently, we have shown that solid lipid Cur particles (SLCPs have greater bioavailability and brain tissue penetration. The present study compares the efficiency of cell death by Cur and/or SLCPs in cultured GBM cells derived from human (U-87MG and mouse (GL261 tissues. Several cell viability and cell death assays and marker proteins (MTT assay, annexin-V staining, TUNEL staining, comet assay, DNA gel electrophoresis, and Western blot were investigated following the treatment of Cur and/or SLCP (25 μM for 24–72 h. Relative to Cur, the use of SLCP increased cell death and DNA fragmentation, produced longer DNA tails, and induced more fragmented nuclear lobes. In addition, cultured GBM cells had increased levels of caspase-3, Bax, and p53, with decreases in Bcl2, c-Myc, and both total Akt, as well as phosphorylated Akt, when SLCP, rather Cur, was used. Our in vitro work suggests that the use of SLCP may be a promising strategy for reversing or preventing GBM growth, as compared to using Cur.

  20. Stem Cell Technology for (Epi)genetic Brain Disorders.

    Science.gov (United States)

    Riemens, Renzo J M; Soares, Edilene S; Esteller, Manel; Delgado-Morales, Raul

    2017-01-01

    Despite the enormous efforts of the scientific community over the years, effective therapeutics for many (epi)genetic brain disorders remain unidentified. The common and persistent failures to translate preclinical findings into clinical success are partially attributed to the limited efficiency of current disease models. Although animal and cellular models have substantially improved our knowledge of the pathological processes involved in these disorders, human brain research has generally been hampered by a lack of satisfactory humanized model systems. This, together with our incomplete knowledge of the multifactorial causes in the majority of these disorders, as well as a thorough understanding of associated (epi)genetic alterations, has been impeding progress in gaining more mechanistic insights from translational studies. Over the last years, however, stem cell technology has been offering an alternative approach to study and treat human brain disorders. Owing to this technology, we are now able to obtain a theoretically inexhaustible source of human neural cells and precursors in vitro that offer a platform for disease modeling and the establishment of therapeutic interventions. In addition to the potential to increase our general understanding of how (epi)genetic alterations contribute to the pathology of brain disorders, stem cells and derivatives allow for high-throughput drugs and toxicity testing, and provide a cell source for transplant therapies in regenerative medicine. In the current chapter, we will demonstrate the validity of human stem cell-based models and address the utility of other stem cell-based applications for several human brain disorders with multifactorial and (epi)genetic bases, including Parkinson's disease (PD), Alzheimer's disease (AD), fragile X syndrome (FXS), Angelman syndrome (AS), Prader-Willi syndrome (PWS), and Rett syndrome (RTT).

  1. Urokinase production by electrophoretically separated cultured human embryonic kidney cells

    Science.gov (United States)

    Kunze, M. E.; Plank, L. D.; Giranda, V.; Sedor, K.; Todd, P. W.

    1985-01-01

    Urokinase is a plasminogen activator found in urine. Relatively pure preparations have been tested in Europe, Japan and the United States for the treatment of deep vein thrombosis and other dangerous blood clots. Human embryonic kidney cell cultures have been found to produce urokinase at much higher concentrations, but less than 5% of the cells in typical cultures are producers. Since human diploid cells become senescent in culture the selection of clones derived from single cells will not provide enough material to be useful, so a bulk purification method is needed for the isolation of urokinase producing cell populations. Preparative cell electrophoresis was chosen as the method, since evidence exists that human embryonic cell cultures are richly heterogeneous with respect to electrophoretic mobility, and preliminary electrophoretic separations on the Apollo-Soyuz space flight produced cell populations that were rich in urokinase production. Similarly, erythropoietin is useful in the treatment of certain anemias and is a kidney cell duct, and electrophoretically enriched cell populations producing this product have been reported. Thus, there is a clear need for diploid human cells that produce these products, and there is evidence that such cells should be separable by free-flow cell electrophoresis.

  2. Controlling the diversity of cell populations in a stem cell culture

    NARCIS (Netherlands)

    Heo, Inha; Clevers, Hans

    2015-01-01

    Culturing intestinal stem cells into 3D organoids results in heterogeneous cell populations, reflecting the in vivo cell type diversity. In a recent paper published in Nature, Wang et al. established a culture condition for a highly homogeneous population of intestinal stem cells.

  3. Systems biology: From the cell to the brain

    Indian Academy of Sciences (India)

    Systems biology: From the cell to the brain. SITABHRA SINHA. 1,∗. , T JESAN. 2 and NIVEDITA CHATTERJEE. 3. 1. The Institute of Mathematical Sciences, CIT Campus, Taramani, Chennai 600 113, India. 2. Health Physics Division, Bhabha Atomic Research Centre, Kalpakkam 603 201, India. 3. Vision Research ...

  4. Characterization of Cancer Stem Cells in Patients with Brain ...

    African Journals Online (AJOL)

    Brain tumor stem cells have been found to express a variety of markers including Nestin, which can be potentially used as therapeutic targets. Dysregulation of the intermediate filament protein Nestin, the tumor-suppressor gene TP53, and Ki67 labeling index are implicated in glioma genesis and therapeutic resistance.

  5. Vascular Cell Senescence Contributes to Blood-Brain Barrier Breakdown

    NARCIS (Netherlands)

    Yamazaki, Y.; Baker, D.J.; Tachibana, M.; Liu, C.C.; Deursen, J.M.A. van; Brott, T.G.; Bu, G.; Kanekiyo, T.

    2016-01-01

    BACKGROUND AND PURPOSE: Age-related changes in the cerebrovasculature, including blood-brain barrier (BBB) disruption, are emerging as potential risks for diverse neurological conditions. Because the accumulation of senescent cells in tissues is increasingly recognized as a critical step leading to

  6. Affinity (tropism) of caprine arthritis encephalitis virus for brain cells

    African Journals Online (AJOL)

    STORAGESEVER

    2008-11-19

    Nov 19, 2008 ... Full Length Research Paper. Affinity (tropism) of caprine arthritis encephalitis virus for brain cells. Adebayo, I. A.1*, Awoniyi, T. A. M. 1 and Olaleye, O. D.2. 1Department of Animal Production and Health, Animal Parasitology and Microbiology Research Unit, Federal University of Technology, P M B 704, ...

  7. The ultrastructure of separated and cultured cell of Porphyra yezoensis

    Science.gov (United States)

    Mei, Jun-Xue; Fei, Xiu-Geng

    2001-03-01

    There are many reports that cells (protoplasts) separated from the thallus of Porphyra by enzyme can develop to normal leafy thalli in the same way as monospores. But there are few investigations on the subcellular structure of the isolated vegetative cell for comparison with the subcellular structure of monospores. To clarify whether the separated and cultured cells undergo the same or similar ultrastructure changes during culture and germination as monospores undergo in their formation and germination, we observed their ultrastructure, compared them with those of the monospore and found that the ultrastructure of separated and cultured cells did not have the characteristic feature as that of monospore formation, such as production of small and large fibrous vesicles, but was accompanied by vacuolation and starch mobilization like that in monospore germination. The paper also discusses the relations between monospores and separated and cultured cells.

  8. Cell death in the injured brain: roles of metallothioneins

    DEFF Research Database (Denmark)

    Pedersen, Mie Ø; Larsen, Agnete; Stoltenberg, Meredin

    2009-01-01

    oxygen species (ROS). ROS promote oxidative stress, which leads to neurodegeneration and ultimately results in programmed cell death (secondary injury). Since this delayed, secondary tissue loss occurs days to months following the primary injury it provides a therapeutic window where potential......In traumatic brain injury (TBI), the primary, irreversible damage associated with the moment of impact consists of cells dying from necrosis. This contributes to fuelling a chronic central nervous system (CNS) inflammation with increased formation of proinflammatory cytokines, enzymes and reactive...

  9. Monitoring of cell cultures with LTCC microelectrode array.

    Science.gov (United States)

    Ciosek, P; Zawadzki, K; Łopacińska, J; Skolimowski, M; Bembnowicz, P; Golonka, L J; Brzózka, Z; Wróblewski, W

    2009-04-01

    Monitoring of cell cultures in microbioreactors is a crucial task in cell bioassays and toxicological tests. In this work a novel tool based on a miniaturized sensor array fabricated using low-temperature cofired ceramics (LTCC) technology is presented. The developed device is applied to the monitoring of cell-culture media change, detection of the growth of various species, and in toxicological studies performed with the use of cells. Noninvasive monitoring performed with the LTCC microelectrode array can be applied for future cell-engineering purposes.

  10. Nylon-3 polymers that enable selective culture of endothelial cells.

    Science.gov (United States)

    Liu, Runhui; Chen, Xinyu; Gellman, Samuel H; Masters, Kristyn S

    2013-11-06

    Substrates that selectively encourage the growth of specific cell types are valuable for the engineering of complex tissues. Some cell-selective peptides have been identified from extracellular matrix proteins; these peptides have proven useful for biomaterials-based approaches to tissue repair or regeneration. However, there are very few examples of synthetic materials that display selectivity in supporting cell growth. We describe nylon-3 polymers that support in vitro culture of endothelial cells but do not support the culture of smooth muscle cells or fibroblasts. These materials may be promising for vascular biomaterials applications.

  11. Growth of melanocytes in human epidermal cell cultures

    International Nuclear Information System (INIS)

    Staiano-Coico, L.; Hefton, J.M.; Amadeo, C.; Pagan-Charry, I.; Madden, M.R.; Cardon-Cardo, C.

    1990-01-01

    Epidermal cell cultures were grown in keratinocyte-conditioned medium for use as burn wound grafts; the melanocyte composition of the grafts was studied under a variety of conditions. Melanocytes were identified by immunohistochemistry based on a monoclonal antibody (MEL-5) that has previously been shown to react specifically with melanocytes. During the first 7 days of growth in primary culture, the total number of melanocytes in the epidermal cultures decreased to 10% of the number present in normal skin. Beginning on day 2 of culture, bipolar melanocytes were present at a mean cell density of 116 +/- 2/mm2; the keratinocyte to melanocyte ratio was preserved during further primary culture and through three subpassages. Moreover, exposure of cultures to mild UVB irradiation stimulated the melanocytes to proliferate, suggesting that the melanocytes growing in culture maintained their responsiveness to external stimuli. When the sheets of cultured cells were enzymatically detached from the plastic culture flasks before grafting, melanocytes remained in the basal layer of cells as part of the graft applied to the patient

  12. Neocortical glial cell numbers in human brains

    DEFF Research Database (Denmark)

    Pelvig, D.P.; Pakkenberg, H.; Stark, A.K.

    2008-01-01

    and neurons and counting were done in each of the four lobes. The study showed that the different subpopulations of glial cells behave differently as a function of age; the number of oligodendrocytes showed a significant 27% decrease over adult life and a strong correlation to the total number of neurons...... while the total astrocyte number is constant through life; finally males have a 28% higher number of neocortical glial cells and a 19% higher neocortical neuron number than females. The overall total number of neocortical neurons and glial cells was 49.3 billion in females and 65.2 billion in males......, a difference of 24% with a high biological variance. These numbers can serve as reference values in quantitative studies of the human neocortex. (C) 2007 Elsevier Inc. All rights reserved Udgivelsesdato: 2008/11...

  13. Surface modified alginate microcapsules for 3D cell culture

    Science.gov (United States)

    Chen, Yi-Wen; Kuo, Chiung Wen; Chueh, Di-Yen; Chen, Peilin

    2016-06-01

    Culture as three dimensional cell aggregates or spheroids can offer an ideal platform for tissue engineering applications and for pharmaceutical screening. Such 3D culture models, however, may suffer from the problems such as immune response and ineffective and cumbersome culture. This paper describes a simple method for producing microcapsules with alginate cores and a thin shell of poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) to encapsulate mouse induced pluripotent stem (miPS) cells, generating a non-fouling surface as an effective immunoisolation barrier. We demonstrated the trapping of the alginate microcapsules in a microwell array for the continuous observation and culture of a large number of encapsulated miPS cells in parallel. miPS cells cultured in the microcapsules survived well and proliferated to form a single cell aggregate. Droplet formation of monodisperse microcapsules with controlled size combined with flow cytometry provided an efficient way to quantitatively analyze the growth of encapsulated cells in a high-throughput manner. The simple and cost-effective coating technique employed to produce the core-shell microcapsules could be used in the emerging field of cell therapy. The microwell array would provide a convenient, user friendly and high-throughput platform for long-term cell culture and monitoring.

  14. Stimulation and support of haemopoietic stem cell proliferation by irradiated stroma cell colonies in bone marrow cell culture in vitro

    International Nuclear Information System (INIS)

    Mori, K.J.; Izumi, Hiroko; Seto, Akira

    1981-01-01

    A culture system was established in which haemopoietic stem cells can undergo a recovery proliferation after a depletion of the stem cells, completely in vitro. To elucidate the source of the stimulatory factors, normal bone marrow cells were overlayed on top of the irradiated adherent 'stromal' cell colonies in the bone marrow cell culture. This stimulated the proliferation of haemopoietic stem cells in the cultured cells in suspension. The present results indicate that the stromal cells produce factors which stimulate stem cell proliferation. Whether the stimulation is evoked by direct cell-cell interactions or by humoral factors is as yet to be studied. (author)

  15. Immunodissection and culture of rabbit cortical collecting tubule cells

    International Nuclear Information System (INIS)

    Spielman, W.S.; Sonnenburg, W.K.; Allen, M.L.; Arend, L.J.; Gerozissis, K.; Smith, W.L.

    1986-01-01

    A mouse monoclonal antibody designated IgG 3 (rct-30) has been prepared that reacts specifically with an antigen on the surface of all cells comprising the cortical and medullary rabbit renal collecting tubule including the arcades. Plastic culture dishes coated with IgG 3 (rct-30) were used to isolate collecting tubule cells from collagenase dispersions of rabbit renal cortical cells by immunoadsorption. Typically, 10 6 rabbit cortical collecting tubule (RCCT) cells were obtained from 5 g of renal cortex (2 kidneys). Between 20 and 30% of the RCCT cells were reactive with peanut lectin suggesting that RCCT cells are a mixture of principal and intercalated cells. Approximately 10 7 RCCT cells were obtained after 4 to 5 days in primary culture. Moreover, RCCT cells continued to proliferate after passaging with a doubling time of ∼32 h. RCCT cells passaged once and then cultured 4-5 days were found 1) to synthesize cAMP in response to arginine vasopressin (AVP), prostaglandin E 2 (PGE 2 ), isoproterenol, and parathyroid hormone, but not calcitonin, prostaglandin D 2 , or prostaglandin I, and 2) to release PGE 2 in response to bradykinin but not arginine vasopressin or isoproterenol. The results indicate that cultured RCCT cells retain many of the hormonal, histochemical, and morphological properties expected for a mixture of principal and intercalated rabbit cortical collecting tubule epithelia. RCCT cells should prove useful both for studying hormonal interactions in the cortical collecting tubule and as a starting population for isolating intercalated collecting tubule epithelia

  16. In vitro production of azadirachtin from cell suspension cultures of ...

    Indian Academy of Sciences (India)

    PRAKASH KUMAR G

    to affect the growth and metabolism of cultured cells, and have been studied extensively in different species in .... Specific growth rate of neem cell suspensions in altered nitrate: ammonium ratio. MS, Murashige and Skoog medium; MS medium with .... Stimulated caffeine production has been reported in Coffea arabica cell ...

  17. Protein biosynthesis in cultured human hair follicle cells.

    Science.gov (United States)

    Weterings, P J; Vermorken, A J; Bloemendal, H

    1980-10-31

    A new technique has been used for culturing human keratinocytes. The cells grow on the basement membrane-like capsules of bovine lenses. Lens cells were removed from the capsules by rigid trypsinization. In order to exclude any contamination with remaining living cells the isolated capsules were irradiated with X-rays at a dose of 10,000 rad. In this way human epithelial cells can be brought in culture from individual hair follicles. Since feeder cells are not used in this culture technique, the biosynthesis of keratinocyte proteins can be studied in these cultures. The newly synthesized proteins can be separated into a water-soluble, a urea-soluble, and a urea-insoluble fraction. Product analysis has been performed on the first two fractions revealing protein patterns identical to those of intact hair follicles. Product analysis of the urea-soluble fractions of microdissected hair follicles shows that the protein pattern of the cultured keratinocytes resembles the protein pattern of the hair follicle sheath. Studies on the metabolism of benzo(a)pyrene revealed that the enzyme aryl hydrocarbon hydroxylase (AHH) is present in cultured hair follicle cells. A possible use of our culture system for eventual detection of inherited predisposition for smoking-dependent lung cancer is discussed.

  18. Cell proliferation and cell death are disturbed during prenatal and postnatal brain development after uranium exposure.

    Science.gov (United States)

    Legrand, M; Elie, C; Stefani, J; N Florès; Culeux, C; Delissen, O; Ibanez, C; Lestaevel, P; Eriksson, P; Dinocourt, C

    2016-01-01

    The developing brain is more susceptible to neurotoxic compounds than adult brain. It is also well known that disturbances during brain development cause neurological disorders in adulthood. The brain is known to be a target organ of uranium (U) exposure and previous studies have noted that internal U contamination of adult rats induces behavioral disorders as well as affects neurochemistry and neurophysiological properties. In this study, we investigated whether depleted uranium (DU) exposure affects neurogenesis during prenatal and postnatal brain development. We examined the structural morphology of the brain, cell death and finally cell proliferation in animals exposed to DU during gestation and lactation compared to control animals. Our results showed that DU decreases cell death in the cortical neuroepithelium of gestational day (GD) 13 embryos exposed at 40mg/L and 120mg/L and of GD18 fetuses exposed at 120mg/L without modification of the number of apoptotic cells. Cell proliferation analysis showed an increase of BrdU labeling in the dentate neuroepithelium of fetuses from GD18 at 120mg/L. Postnatally, cell death is increased in the dentate gyrus of postnatal day (PND) 0 and PND5 exposed pups at 120mg/L and is associated with an increase of apoptotic cell number only at PND5. Finally, a decrease in dividing cells is observed in the dentate gyrus of PND21 rats developmentally exposed to 120mg/L DU, but not at PND0 and PND5. These results show that DU exposure during brain development causes opposite effects on cell proliferation and cell death processes between prenatal and postnatal development mainly at the highest dose. Although these modifications do not have a major impact in brain morphology, they could affect the next steps of neurogenesis and thus might disrupt the fine organization of the neuronal network. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. Rapid metabolism of exogenous angiotensin II by catecholaminergic neuronal cells in culture media.

    Science.gov (United States)

    Basu, Urmi; Seravalli, Javier; Madayiputhiya, Nandakumar; Adamec, Jiri; Case, Adam J; Zimmerman, Matthew C

    2015-02-01

    Angiotensin II (AngII) acts on central neurons to increase neuronal firing and induce sympathoexcitation, which contribute to the pathogenesis of cardiovascular diseases including hypertension and heart failure. Numerous studies have examined the precise AngII-induced intraneuronal signaling mechanism in an attempt to identify new therapeutic targets for these diseases. Considering the technical challenges in studying specific intraneuronal signaling pathways in vivo, especially in the cardiovascular control brain regions, most studies have relied on neuronal cell culture models. However, there are numerous limitations in using cell culture models to study AngII intraneuronal signaling, including the lack of evidence indicating the stability of AngII in culture media. Herein, we tested the hypothesis that exogenous AngII is rapidly metabolized in neuronal cell culture media. Using liquid chromatography-tandem mass spectrometry, we measured levels of AngII and its metabolites, Ang III, Ang IV, and Ang-1-7, in neuronal cell culture media after administration of exogenous AngII (100 nmol/L) to a neuronal cell culture model (CATH.a neurons). AngII levels rapidly declined in the media, returning to near baseline levels within 3 h of administration. Additionally, levels of Ang III and Ang-1-7 acutely increased, while levels of Ang IV remained unchanged. Replenishing the media with exogenous AngII every 3 h for 24 h resulted in a consistent and significant increase in AngII levels for the duration of the treatment period. These data indicate that AngII is rapidly metabolized in neuronal cell culture media, and replenishing the media at least every 3 h is needed to sustain chronically elevated levels. © 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.

  20. Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells.

    Science.gov (United States)

    Nielsen, Simone S E; Siupka, Piotr; Georgian, Ana; Preston, Jane E; Tóth, Andrea E; Yusof, Siti R; Abbott, N Joan; Nielsen, Morten S

    2017-09-24

    The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) models based on pBECs in mono-culture (MC), MC with astrocyte-conditioned medium (ACM), and non-contact co-culture (NCC) with astrocytes of porcine or rat origin. pBECs were isolated and cultured from fragments of capillaries from the brain cortices of domestic pigs 5-6 months old. These fragments were purified by careful removal of meninges, isolation and homogenization of grey matter, filtration, enzymatic digestion, and centrifugation. To further eliminate contaminating cells, the capillary fragments were cultured with puromycin-containing medium. When 60-95% confluent, pBECs growing from the capillary fragments were passaged to permeable membrane filter inserts and established in the models. To increase barrier tightness and BBB characteristic phenotype of pBECs, the cells were treated with the following differentiation factors: membrane permeant 8-CPT-cAMP (here abbreviated cAMP), hydrocortisone, and a phosphodiesterase inhibitor, RO-20-1724 (RO). The procedure was carried out over a period of 9-11 days, and when establishing the NCC model, the astrocytes were cultured 2-8 weeks in advance. Adherence to the described procedures in the protocol has allowed the establishment of endothelial layers with highly restricted paracellular permeability, with the NCC model showing an average transendothelial electrical resistance (TEER) of 1249 ± 80 Ω cm 2 , and paracellular permeability (Papp) for Lucifer Yellow of 0.90 10 -6 ± 0.13 10 -6 cm sec -1 (mean ± SEM, n=55). Further evaluation of this pBEC phenotype showed good expression of the tight junctional proteins claudin 5, ZO-1, occludin and adherens junction protein p120 catenin. The model presented can be used for a range of studies of the BBB in health and disease and, with the highly restrictive paracellular permeability, this model is suitable for studies

  1. Regulation of apoptosis and cell cycle in irradiated mouse brain

    Energy Technology Data Exchange (ETDEWEB)

    Oh, Won Yong; Song, Mi Hee; Hung, Eun Ji; Seong, Jin Sil; Suh, Chang Ok [College of Medicine, Yonsei Univ., Seoul (Korea, Republic of)

    2001-06-01

    To investigate the regulation of apoptosis and cell cycle in mouse brain irradiation. 8-week old male mice, C57B 1/6J were given whole body {gamma} -radiation with a single dose of 25 Gy using Cobalt 60 irradiator. At different times 1, 2, 4, 8 and 24hr after irradiation, mice were killed and brain tissues were collected. Apoptotic cells were scored by TUNEL assay. Expression of p53, Bcl-2, and Bax and cell cycle regulating molecules; cyclins BI, D1, E and cdk2, cdk4, p34{sup cdc2} were analysed by Western blotting. Cell cycle was analysed by flow cytometry. The peak of radiation induced apoptosis is shown at 8 hour after radiation. With a single 25 Gy irradiation, the peak of apoptotic index in C57B1/6J is 24.0{+-}0.25 (p<0.05) at 8 hour after radiation. Radiation upregulated the expression of p53/tubulin, Bax/tubulin, and Bcl-2/tubulin with 1.3, 1.1 and 1.45 fold increase, respectively were shown at the peak level at 8 hour after radiation. The levels of cell cycle regulating molecules after radiation are not changed significantly except cyclin D1 with 1.3 fold increase. Fractions of Go-G 1, G2-M and S phase in the cell cycle does not specific changes by time. In mouse brain tissue, radiation induced apoptosis is particularly shown in a specific area, subependyma. These results and lack of radiation induced changes in cell cycle offer better understanding of radiation response of normal brain tissue.

  2. Control of fibronectin synthesis by rat granulosa cells in culture

    International Nuclear Information System (INIS)

    Skinner, M.K.; Dorrington, J.H.

    1984-01-01

    The secreted and cellular [ 35 S]methionine-radiolabeled proteins of cultured rat granulosa cells were separated by electrophoresis on sodium dodecylsulfate (SDS) polyacrylamide gradient slab gels. From 24 to 72 h of culture FSH increased the intensity of labeling of most of the secreted proteins. A 220,000-dalton protein, however, increased in intensity only in control cultures and became the major secreted protein after 72 h, comprising 20% of the total radiolabeled proteins. This protein was identified as fibronectin by immunoprecipitation. There was no increase in the secreted or cellular fibronectin in FSH- or testosterone- and insulin-treated cultures. These studies indicate that a component of extracellular matrix is a major secretory product of unstimulated immature granulosa cells. As hormones induce the differentiated functions of granulosa cells in culture, the secretion of fibronectin is inhibited

  3. Characterization of a novel miniature cell culture device

    Science.gov (United States)

    Moore, Sandra K.; Kleis, Stanley J.

    2008-05-01

    Recent advancements in the field of microfluidics have generated much interest in the advent of a miniaturized cell culture device. In this study, we developed a novel miniature culture system (cells, either prokaryotic or eukaryotic in type, for both 1 g and microgravity applications. The miniature culture system may advance the development of microanalytical remote monitoring tools such as biological sentinels, biosensors, and lab-on-a-chip. Integrating the autonomous miniature culture system with a microanalytical device makes a powerful biological tool. Cells can be cultured long-term, harvested, and released directly into an analytical tool without the need for human interaction through fluid dynamic manipulations. This work characterizes the miniature bioreactor system through numerical and experimental proof of concept studies.

  4. Challenges in providing culturally-competent care to patients with metastatic brain tumours and their families.

    Science.gov (United States)

    Longo, Lianne; Slater, Serena

    2014-01-01

    Being diagnosed with a metastatic brain tumour can be devastating as it is characterized by very low cure rates, as well as significant morbidity and mortality. Given the poor life expectancy and progressive disability that ensues, patients and family members experience much turmoil, which includes losses that bring about changes to family roles, routines and relationships. Crisis and conflict are common during such major disruptions to a family system, as individual members attempt to make sense of the illness experience based on cultural and spiritual beliefs, past experiences and personal philosophies. It is imperative health care providers strive towards increased awareness and knowledge of how culture affects the overall experience of illness and death in order to help create a mutually satisfactory care plan. Providing culturally-competent care entails the use of proper communication skills to facilitate the exploration of patient and family perspectives and allows for mutual decision making. A case study will illustrate the challenges encountered in providing culturally-competent care to a woman with brain cancer and her family. As the patient's health declined, the family entered into a state of crisis where communication between family members and health care professionals was strained; leading to conflict and sub-optimal outcomes. This paper will address the ethical dilemma of providing culturally-competent care when a patient's safety is at risk, and the nursing implications of upholding best practices in the context of differing beliefs and priorities.

  5. Purification of cells from fresh human brain tissue: primary human glial cells.

    NARCIS (Netherlands)

    Mizee, Mark R; van der Poel, Marlijn; Huitinga, I.; Huitinga, I.; Webster, M.J.

    2018-01-01

    In order to translate the findings obtained from postmortem brain tissue samples to functional biologic mechanisms of central nervous system disease, it will be necessary to understand how these findings affect the different cell populations in the brain. The acute isolation and analysis of pure

  6. Glial-Restricted Precursors Protect Neonatal Brain Slices from Hypoxic-Ischemic Cell Death Without Direct Tissue Contact.

    Science.gov (United States)

    Sweda, Romy; Phillips, Andre W; Marx, Joel; Johnston, Michael V; Wilson, Mary Ann; Fatemi, Ali

    2016-07-01

    Glial-Restricted Precursors (GRPs) are tripotential progenitors that have been shown to exhibit beneficial effects in several preclinical models of neurological disorders, including neonatal brain injury. The mechanisms of action of these cells, however, require further study, as do clinically relevant questions such as timing and route of cell administration. Here, we explored the effects of GRPs on neonatal hypoxia-ischemia during acute and subacute stages, using an in vitro transwell co-culture system with organotypic brain slices exposed to oxygen-glucose deprivation (OGD). OGD-exposed slices that were then co-cultured with GRPs without direct cell contact had decreased tissue injury and cortical cell death, as evaluated by lactate dehydrogenase (LDH) release and propidium iodide (PI) staining. This effect was more pronounced when cells were added during the subacute phase of the injury. Furthermore, GRPs reduced the amount of glutamate in the slice supernatant and changed the proliferation pattern of endogenous progenitor cells in brain slices. In summary, we show that GRPs exert a neuroprotective effect on neonatal hypoxia-ischemia without the need for direct cell-cell contact, thus confirming the rising view that beneficial actions of stem cells are more likely attributable to trophic or immunomodulatory support rather than to long-term integration.

  7. Expression and deposition of basement membrane proteins by brain capillary endothelial cells in a primary murine model of the blood-brain barrier

    DEFF Research Database (Denmark)

    Thomsen, Maj Schneider; Birkelund, Svend; Larsen, Annette Burkhart

    2016-01-01

    of the present study was to create four different in vitro constructs of the murine BBB to characterise if the expression and secretion of basement membrane proteins by the murine brain capillary endothelial cells (mBCECs) was affected by co-culturing with pericytes, mixed glial cells, or both. Primary m...... membrane, and astrocyte endfeet. To study the interaction of the different cells of the BBB, construction of in vitro BBB models is valuable. However, the modulation and contribution of the cells of the BBB to the synthesis of basement membrane proteins in vitro is not fully elaborated. Thus, the aim......, and immunofluorescent labelling were used. The mBCECs were found to express major basement membrane proteins in vitro and increased expression of laminin α5 and collagen IV α1 was correlated to the addition of BBB inducing factors (hydrocortisone, Ro20-1724, pCPT-cAMP). Co-culturing of the mBCECs with pericytes, mixed...

  8. Radiosensitivity of cultured insect cells: II. Diptera

    Energy Technology Data Exchange (ETDEWEB)

    Koval, T.M.

    1983-10-01

    The radiosensitivity of five dipteran cell lines representing three mosquito genera and one fruit fly genus were examined. These lines are: (1) ATC-10, Aedes aegypti; (2) RU-TAE-14, Toxorhynchites amboinensis; (3) RU-ASE-2A, Anopheles stephensi; (4) WR69-DM-1, Drosophila melanogaster; and (5) WR69-DM-2, Drosophila melanogaster. Population doubling times for these lines range from approximately 16 to 48 hr. Diploid chromosome numbers are six for the mosquito cells and eight for the fruit fly cells D/sub 0/ values are 5.1 and 6.5 Gy for the Drosophila cell lines and 3.6, 6.2, and 10.2 Gy for the mosquito cell lines. The results of this study demonstrate that dipteran insect cells are a few times more resistant to radiation than mammalian cells, but not nearly as radioresistant as lepidopteran cells.

  9. Radiosensitivity of cultured insect cells: II. Diptera

    International Nuclear Information System (INIS)

    Koval, T.M.

    1983-01-01

    The radiosensitivity of five dipteran cell lines representing three mosquito genera and one fruit fly genus were examined. These lines are: (1) ATC-10, Aedes aegypti; (2) RU-TAE-14, Toxorhynchites amboinensis; (3) RU-ASE-2A, Anopheles stephensi; (4) WR69-DM-1, Drosophila melanogaster; and (5) WR69-DM-2, Drosophila melanogaster. Population doubling times for these lines range from approximately 16 to 48 hr. Diploid chromosome numbers are six for the mosquito cells and eight for the fruit fly cells D 0 values are 5.1 and 6.5 Gy for the Drosophila cell lines and 3.6, 6.2, and 10.2 Gy for the mosquito cell lines. The results of this study demonstrate that dipteran insect cells are a few times more resistant to radiation than mammalian cells, but not nearly as radioresistant as lepidopteran cells

  10. Quantitative volumetric Raman imaging of three dimensional cell cultures

    KAUST Repository

    Kallepitis, Charalambos

    2017-03-22

    The ability to simultaneously image multiple biomolecules in biologically relevant three-dimensional (3D) cell culture environments would contribute greatly to the understanding of complex cellular mechanisms and cell–material interactions. Here, we present a computational framework for label-free quantitative volumetric Raman imaging (qVRI). We apply qVRI to a selection of biological systems: human pluripotent stem cells with their cardiac derivatives, monocytes and monocyte-derived macrophages in conventional cell culture systems and mesenchymal stem cells inside biomimetic hydrogels that supplied a 3D cell culture environment. We demonstrate visualization and quantification of fine details in cell shape, cytoplasm, nucleus, lipid bodies and cytoskeletal structures in 3D with unprecedented biomolecular specificity for vibrational microspectroscopy.

  11. Generation of a patterned co-culture system composed of adherent cells and immobilized nonadherent cells.

    Science.gov (United States)

    Yamazoe, Hironori; Ichikawa, Takashi; Hagihara, Yoshihisa; Iwasaki, Yasuhiko

    2016-02-01

    Patterned co-culture is a promising technique used for fundamental investigation of cell-cell communication and tissue engineering approaches. However, conventional methods are inapplicable to nonadherent cells. In this study, we aimed to establish a patterned co-culture system composed of adherent and nonadherent cells. Nonadherent cells were immobilized on a substrate using a cell membrane anchoring reagent conjugated to a protein, in order to incorporate them into the co-culture system. Cross-linked albumin film, which has unique surface properties capable of regulating protein adsorption, was used to control their spatial localization. The utility of our approach was demonstrated through the fabrication of a patterned co-culture consisting of micropatterned neuroblastoma cells surrounded by immobilized myeloid cells. Furthermore, we also created a co-culture system composed of cancer cells and immobilized monocytes. We observed that monocytes enhanced the drug sensitivity of cancer cells and its influence was limited to cancer cells located near the monocytes. Therefore, the incorporation of nonadherent cells into a patterned co-culture system is useful for creating culture systems containing immune cells, as well as investigating the influence of these immune cells on cancer drug sensitivity. Various methods have been proposed for creating patterned co-culture systems, in which multiple cell types are attached to a substrate with a desired pattern. However, conventional methods, including our previous report published in Acta Biomaterialia (2010, 6, 526-533), are unsuitable for nonadherent cells. Here, we developed a novel method that incorporates nonadherent cells into the co-culture system, which allows us to precisely manipulate and study microenvironments containing nonadherent and adherent cells. Using this technique, we demonstrated that monocytes (nonadherent cells) could enhance the drug sensitivity of cancer cells and that their influence had a

  12. Human hematopoietic cell culture, transduction, and analyses

    DEFF Research Database (Denmark)

    Bonde, Jesper; Wirthlin, Louisa; Kohn, Donald B

    2008-01-01

    This unit provides methods for introducing genes into human hematopoietic progenitor cells. The Basic Protocol describes isolation of CD34(+) cells, transduction of these cells with a retroviral vector on fibronectin-coated plates, assaying the efficiency of transduction, and establishing long-te...

  13. Exposure to Music Alters Cell Viability and Cell Motility of Human Nonauditory Cells in Culture.

    Science.gov (United States)

    Lestard, Nathalia R; Capella, Marcia A M

    2016-01-01

    Although music is part of virtually all cultures in the world, little is known about how it affects us. Since the beginning of this century several studies suggested that the response to music, and to sound in general, is complex and might not be exclusively due to emotion, given that cell types other than auditory hair cells can also directly react to audible sound. The present study was designed to better understand the direct effects of acoustic vibrations, in the form of music, in human cells in culture. Our results suggest that the mechanisms of cell growth arrest and/or cell death induced by acoustic vibrations are similar for auditory and nonauditory cells.

  14. Establishment of sorghum cell suspension culture system for ...

    African Journals Online (AJOL)

    Total soluble proteins (TSP) and culture filtrate (CF) proteins were extracted from the cell culture system and solubilised in urea buffer (9 M urea, 2 M thiourea and 4% CHAPS). Both onedimensional (1D) and two-dimensional (2D) gel analysis of these two proteomes show that the TSP and CF proteomes have different ...

  15. Free-energy carriers in human cultured muscle cells

    NARCIS (Netherlands)

    Bolhuis, P. A.; de Zwart, H. J.; Ponne, N. J.; de Jong, J. M.

    1985-01-01

    Creatine phosphate (CrP), adenosine triphosphate (ATP), creatine kinase (CK), adenylate kinase (AK), protein, and DNA were quantified in human muscle cell cultures undergoing transition from dividing myoblasts to multinucleate myotubes. CrP is negligible in cultures grown in commonly applied media

  16. Enhancement of Diosgenin Production in Plantlet and Cell Cultures ...

    African Journals Online (AJOL)

    Enhancement of Diosgenin Production in Plantlet and Cell Cultures of Dioscorea zingiberensis by Palmarumycin C13 from the Endophytic fungus, Berkleasmium sp. Dzf12. Y Mou, K Zhou, D Xu, R Yu, J Li, C Yin, L Zhou ...

  17. Impact of cell culture on recombinant monoclonal antibody product heterogeneity.

    Science.gov (United States)

    Liu, Hongcheng; Nowak, Christine; Shao, Mei; Ponniah, Gomathinayagam; Neill, Alyssa

    2016-09-01

    Recombinant monoclonal antibodies are commonly expressed in mammalian cell culture and purified by several steps of filtration and chromatography. The resulting high purity bulk drug substance still contains product variants differing in properties such as charge and size. Posttranslational modifications and degradations occurring during cell culture are the major sources of heterogeneity in bulk drug substance of recombinant monoclonal antibodies. The focus of the current review is the impact of cell culture conditions on the types and levels of various modifications and degradations of recombinant monoclonal antibodies. Understanding the relationship between cell culture and product variants can help to make consistently safe and efficacious products. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1103-1112, 2016. © 2016 American Institute of Chemical Engineers.

  18. Viral risk mitigation for Mammalian cell culture media.

    Science.gov (United States)

    Weaver, Bob; Rosenthal, Scott

    2010-01-01

    Adventitious viral contamination in mammalian cell culture manufacturing facilities can lead to loss of product due to regulatory concerns regarding potential health risks. These events can also result in manufacturing shutdowns for extended periods of time. Numerous measures are currently taken to minimize these risks. Nonetheless, raw materials remain a high-risk entry point for viral contamination of mammalian cell cultures. Two virucidal technologies, ultraviolet radiation in the C band and high-temperature short-time pasteurization, were tested for the treatment of mammalian cell culture media. The results demonstrated no impact to the cell culture process or the quality of the products produced at the chosen dosage while providing robust viral protection.

  19. Elicitation of Diacetylenic Compounds in Suspension Cultured Cells of Eggplant

    Science.gov (United States)

    Imoto, Setsuko; Ohta, Yoshimoto

    1988-01-01

    Induction of stress metabolites in the suspension cultured cells of eggplant (Solanum melongena L.) was examined. When autoclaved RNase A or nigeran, both of which are nonspecific phytoalexin elicitors in bean cells, were added to the cell culture of eggplant, greatly enhanced levels of three compounds were observed. One of them was cis-pentadeca-6-ene-1,3-diyne-5,15-diol, a novel diacetylenic compound. This compound has considerable fungitoxic activity. Also identified was falcarindiol, another fungitoxic diacetylenic compound previously reported as one of the phytoalexins in infected tomato fruits and leaves. Elicited compounds preferentially accumulated in the culture medium rather than in the cells and decreased to original levels during prolonged culturing. The elicitation of these compounds was closely correlated with cellular damage in terms of the decrease of growth rate and was inhibited by 10 micromolar cycloheximide. PMID:16665862

  20. Cell/Tissue Culture Radiation Exposure Facility, Phase I

    Data.gov (United States)

    National Aeronautics and Space Administration — We propose to develop a Cell/Tissue Culture Radiation Exposure Facility (CTC-REF) to enable radiobiologists to investigate the real-time radiation effects on...

  1. Stereotaxic implantation of dispersed cell suspensions into brain. A systematic appraisal of cell placement and survival

    International Nuclear Information System (INIS)

    Plunkett, R.J.; Weber, R.J.; Oldfield, E.H.

    1988-01-01

    The application of several recent advances in cell biology, brain implantation, and cell-mediated tumor immunotherapy requires successful and reproducible placement of viable cell suspensions into brain. Stereotaxic implantation is being used to inject cytotoxic lymphocytes into gliomas and to replace dopaminergic cells in parkinsonian models. Systematic assessment of the factors that influence success in implantation of cell suspensions into solid tissues is needed. A model was developed for investigation of stereotaxic implantation using radiolabeled rat lymphokine-activated killer (LAK) cells. Anesthetized rats received microliter injections of cell suspension into the right caudate nucleus. The injection volume, cell concentration, infusion rate, and needle size were varied systematically. The animals were sacrificed 1 hour after injection; the brain was removed and sectioned, and the radioactivity was counted. Three aliquots of the suspension were injected into counting tubes for control analysis. Recovery of radioactivity was expressed as the percent of mean counts per minute (cpm) in the right frontal lobe/mean cpm in the three control tubes. To assess the viability of implanted cells, the right frontal region was mechanically dissociated in media and centrifuged, and the pellet and supernatant were counted. By using small needles and slow infusion of volumes of 10 microliters or less, 85% to 90% of the radioactivity was recovered in the caudate nucleus. At least half of the implanted cells were viable. Consistent, accurate implantation of dispersed cells into brain over a range of volumes, cell concentrations, infusion rates, and needle sizes was achieved

  2. Characterization of Tight Junction Proteins in Cultured Human Urothelial Cells

    Science.gov (United States)

    Rickard, Alice; Dorokhov, Nikolay; Ryerse, Jan; Klumpp, David J.; McHowat, Jane

    2010-01-01

    Tight junctions (TJs) are essential for normal function of epithelia, restricting paracellular diffusion and contributing to the maintainance of cell surface polarity. Superficial cells of the urothelium develop TJs, the basis for the paracellular permeability barrier of the bladder against diffusion of urinary solutes. Focusing on the superficial cell layer of stratified cell cultures of an immortalized human ureteral cell line, TEU-2 cells, we have examined the presence of TJ and TJ-associated proteins. TEU-2 cells were treated with calcium chloride and fetal bovine serum culture conditions used to induce stratification that resembles the normal transitional epithelial phenotype. Cultures were examined for TJ and TJ-associated proteins by confocal immuno-fluorescence microscopy and evaluated for TJ mRNA by reverse transcriptase-polymerase chain reaction (RT- PCR). TEU-2 cultures exhibited immunoreactivity at intercellular margins for claudins 1, 4, 5, 7, 14 and 16 whereas claudins 2, 8 and 12 were intracellular. RT-PCR corroborated the presence of these claudins at the mRNA level. The TJ-associated proteins occludin, JAM-1, and zonula occludens (ZO-1, ZO-2 and ZO-3) were localized at cell margins. We have found that numerous TJs and TJ-associated proteins are expressed in stratified TEU-2 cultures. Further, we propose TEU-2s provide a useful ureteral model for future studies on the involvement of TJs proteins in the normal and pathological physiology of the human urinary system. PMID:18553212

  3. Cell culture plastics with immobilized interleukin-4 for monocyte differentiation

    DEFF Research Database (Denmark)

    Hansen, Morten; Hjortø, Gertrud Malene; Met, Özcan

    2011-01-01

    Standard cell culture plastic was surface modified by passive adsorption or covalent attachment of interleukin (IL)-4 and investigated for its ability to induce differentiation of human monocytes into mature dendritic cells, a process dose-dependently regulated by IL-4. Covalent attachment of IL-4...... in water instead of phosphate-buffered saline. Passively adsorbed IL-4 was observed to induce differentiation to dendritic cells, but analysis of cell culture supernatants revealed that leakage of IL-4 into solution could account for the differentiation observed. Covalent attachment resulted in bound IL-4...

  4. Gene delivery of the therapeutic polypeptide erythropoietin to primary brain capillary endothelial cells for protein secretion

    DEFF Research Database (Denmark)

    Larsen, Annette Burkhart; Moos, Torben

    2016-01-01

    The potential for treatment of chronic disorders affecting the CNS is complicated by the inability of several drugs to cross the blood-brain barrier (BBB). None-viral gene therapy applied to brain capillary endothelial cells (BCECs) denotes a novel approach to overcome the restraints in this pass......The potential for treatment of chronic disorders affecting the CNS is complicated by the inability of several drugs to cross the blood-brain barrier (BBB). None-viral gene therapy applied to brain capillary endothelial cells (BCECs) denotes a novel approach to overcome the restraints......, however, recently shown that non-viral gene therapy to non-mitotic BCECs cultured in an in-vitro BBB model was possible without disrupting the barrier properties of the BCECs, and that this was as effective as transfection of dividing BCECs. The transfection efficiency is, however, low, questioning...... for its neuroprotective potential, from the BCECs. Our study opens for knowledge on, how non-viral gene therapy to BCECs can lead to protein secretion with the perspective of enabling therapeutic proteins to target neurons inside the CNS otherwise inhibited to access due to the restraints of the BBB...

  5. Domoic acid disrupts the activity and connectivity of neuronal networks in organotypic brain slice cultures.

    Science.gov (United States)

    Hiolski, E M; Ito, S; Beggs, J M; Lefebvre, K A; Litke, A M; Smith, D R

    2016-09-01

    Domoic acid is a neurotoxin produced by algae and is found in seafood during harmful algal blooms. As a glutamate agonist, domoic acid inappropriately stimulates excitatory activity in neurons. At high doses, this leads to seizures and brain lesions, but it is unclear how lower, asymptomatic exposures disrupt neuronal activity. Domoic acid has been detected in an increasing variety of species across a greater geographical range than ever before, making it critical to understand the potential health impacts of low-level exposure on vulnerable marine mammal and human populations. To determine whether prolonged domoic acid exposure altered neuronal activity in hippocampal networks, we used a custom-made 512 multi-electrode array with high spatial and temporal resolution to record extracellular potentials (spikes) in mouse organotypic brain slice cultures. We identified individual neurons based on spike waveform and location, and measured the activity and functional connectivity within the neuronal networks of brain slice cultures. Domoic acid exposure significantly altered neuronal spiking activity patterns, and increased functional connectivity within exposed cultures, in the absence of overt cellular or neuronal toxicity. While the overall spiking activity of neurons in domoic acid-exposed cultures was comparable to controls, exposed neurons spiked significantly more often in bursts. We also identified a subset of neurons that were electrophysiologically silenced in exposed cultures, and putatively identified those neurons as fast-spiking inhibitory neurons. These results provide evidence that domoic acid affects neuronal activity in the absence of cytotoxicity, and suggest that neurodevelopmental exposure to domoic acid may alter neurological function in the absence of clinical symptoms. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. CULTURE OF EMBRYONIC CELLS OF DROSOPHILA MELANOGASTER IN VITRO.

    Science.gov (United States)

    HORIKAWA, M; FOX, A S

    1964-09-25

    Embryonic cells isolated from eggs ofDrosophila melanogasterhave been cultured continuously in a new medium. Generation time for cell division is 30 hours. Chromosome number remains constant for at least 10 days. Cells from embryos of the mutant maroon-like grow at the same rate as those from wild-type embryos, but cells from rosy-2 grow slower and at a lower optimum temperature.

  7. Cell proliferation and radiosensitivity of cow lymphocytes in culture

    International Nuclear Information System (INIS)

    Modave, C.; Fabry, L.; Leonard, A.

    1982-01-01

    The harlequin-staining technique has been used to study, after PHA-stimulation, the cell proliferation of cow lymphocytes in culture and to assess the radiosensitivity in first mitosis cells. At the 48 h fixation time, only 34% of the cells are in first mitosis whereas 55% are already in second and 11% in third mitosis. The exposure of cow lymphocytes to 200 rad X-rays result in the production of 16% dicentric chromosomes in first mitosis cells [fr

  8. Introducing Mammalian Cell Culture and Cell Viability Techniques in the Undergraduate Biology Laboratory.

    Science.gov (United States)

    Bowey-Dellinger, Kristen; Dixon, Luke; Ackerman, Kristin; Vigueira, Cynthia; Suh, Yewseok K; Lyda, Todd; Sapp, Kelli; Grider, Michael; Crater, Dinene; Russell, Travis; Elias, Michael; Coffield, V McNeil; Segarra, Verónica A

    2017-01-01

    Undergraduate students learn about mammalian cell culture applications in introductory biology courses. However, laboratory modules are rarely designed to provide hands-on experience with mammalian cells or teach cell culture techniques, such as trypsinization and cell counting. Students are more likely to learn about cell culture using bacteria or yeast, as they are typically easier to grow, culture, and manipulate given the equipment, tools, and environment of most undergraduate biology laboratories. In contrast, the utilization of mammalian cells requires a dedicated biological safety cabinet and rigorous antiseptic techniques. For this reason, we have devised a laboratory module and method herein that familiarizes students with common cell culture procedures, without the use of a sterile hood or large cell culture facility. Students design and perform a time-efficient inquiry-based cell viability experiment using HeLa cells and tools that are readily available in an undergraduate biology laboratory. Students will become familiar with common techniques such as trypsinizing cells, cell counting with a hemocytometer, performing serial dilutions, and determining cell viability using trypan blue dye. Additionally, students will work with graphing software to analyze their data and think critically about the mechanism of death on a cellular level. Two different adaptations of this inquiry-based lab are presented-one for non-biology majors and one for biology majors. Overall, these laboratories aim to expose students to mammalian cell culture and basic techniques and help them to conceptualize their application in scientific research.

  9. Culturing intestinal stem cells: applications for colorectal cancer research

    Directory of Open Access Journals (Sweden)

    Masayuki eFujii

    2014-06-01

    Full Text Available Recent advance of sequencing technology has revealed genetic alterations in colorectal cancer. The biological function of recurrently mutated genes has been intensively investigated through mouse genetic models and colorectal cancer cell lines. Although these experimental models may not fully reflect biological traits of human intestinal epithelium, they provided insights into the understanding of intestinal stem cell self-renewal, leading to the development of novel human intestinal organoid culture system. Intestinal organoid culture enabled to expand normal or tumor epithelial cells in vitro retaining their stem cell self-renewal and multiple differentiation. Gene manipulation of these cultured cells may provide an attractive tool for investigating genetic events involved in colorectal carcinogenesis.

  10. Production of recombinant proteins in suspension-cultured plant cells.

    Science.gov (United States)

    Plasson, Carole; Michel, Rémy; Lienard, David; Saint-Jore-Dupas, Claude; Sourrouille, Christophe; de March, Ghislaine Grenier; Gomord, Véronique

    2009-01-01

    Plants have emerged in the past decade as a suitable alternative to the current production systems for recombinant pharmaceutical proteins and, today their potential for low-cost production of high quality, much safer and biologically active mammalian proteins is largely documented. Among various plant expression systems being explored, genetically modified suspension-cultured plant cells offer a promising system for production of biopharmaceuticals. Indeed, when compared to other plant-based production platforms that have been explored, suspension-cultured plant cells have the advantage of being totally devoid of problems associated with the vagaries of weather, pest, soil and gene flow in the environment. Because of short growth cycles, the timescale needed for the production of recombinant proteins in plant cell culture can be counted in days or weeks after transformation compared to months needed for the production in transgenic plants. Moreover, recovery and purification of recombinant proteins from plant biomass is an expensive and technically challenging business that may amount to 80-94% of the final product cost. One additional advantage of plant cell culture is that the recombinant protein fused with a signal sequence can be expressed and secreted into the culture medium, and therefore recovered and purified in the absence of large quantities of contaminating proteins. Consequently, the downstream processing of proteins extracted from plant cell culture medium is less expensive, which may/does balance the higher costs of fermentation. When needed for clinical use, recombinant proteins are easily produced in suspension-cultured plant cells under certified, controllable and sterile conditions that offer improved safety and provide advantages for good manufacturing practices and regulatory compliance. In this chapter, we present basic protocols for rapid generation of transgenic suspension-cultured cells of Nicotiana tabacum, Oriza sativa and Arabidopis

  11. Determination of thymidine in serum used for cell culture media

    International Nuclear Information System (INIS)

    Schaer, J.C.; Maurer, U.; Schindler, R.

    1978-01-01

    Thymidine concentrations in serum used for cell culture media were determined with an assay based on isotope dilution. In this assay, incorporation of (3H)-thymidine into DNA of cultured cells was measured in the presence of 5 and 20% serum as a function of the concentration of unlabeled thymidine added to the medium. Thymidine concentrations were measured using horse serum as well as fetal calf serum in the culture media. Dialysis of serum resulted in a reduction of thymidine levels by factors of at least 10

  12. Characterization of cysteinyl-leukotriene formation in primary astroglial cell cultures.

    Science.gov (United States)

    Seregi, A; Simmet, T; Schobert, A; Hertting, G

    1990-03-01

    The formation and composition of cysteinyl-leukotrienes (LT) in primary astroglial cell cultures prepared from newborn rat brain has been studied. Small amounts of cysteinyl-LT determined in terms of LTC4-like material in the supernatants of the cultures, became detectable after stimulation of the cells with 10(-5) M ionophore A23187. Cysteinyl-LT formation increased with time, reaching about 600 pg (mg protein)-1 after 60 min incubation. In contrast, considerable thromboxane (TX) B2 synthesis was found at 5 min following A23187-stimulation (about 30 ng TXB2 (mg protein)-1). The synthesis of cysteinyl-LT was abolished by 5 x 10(-5) M nordihydroguaiaretic acid (NDGA). Irrespective of the duration of incubation, blockage of prostanoid synthesis by 10(-6) M indomethacin did not result in increased cysteinyl-LT production. Reversed phase HPLC combined with radioimmunological detection showed that, after 60 min incubation in the presence of A23187, LTC4 and LTD4 accounted for practically all the LTC4-like immunoreactive material in the supernatants of cell cultures. No significant amounts of LTE4 could be detected. The results show that astrocytes may contribute to brain LTC4 and LTD4 synthesis. However, the cellular site of cerebral LTE4 formation seems to be other than the astroglia.

  13. The effect of tobacco smoke exposure on the generation of reactive oxygen species and cellular membrane damage using co-culture model of blood brain barrier with astrocytes.

    Science.gov (United States)

    Seo, Seung-Beom; Choe, Eun Sang; Kim, Kwang-Sik; Shim, Soon-Mi

    2017-06-01

    Brain tissue is known to be vulnerable to the exposure by tobacco smoke. Tobacco smoke can induce generation of reactive oxygen species (ROS), causing inflammatory activity and blood-brain barrier (BBB) impairment. The aim of the present study was to investigate the effect of tobacco smoke on cell cytotoxicity, generation of ROS, and cellular membrane damage in astrocytes and BBB using a co-culture system. Cell viability of U373MG cells was reduced in a dose-dependent manner, ranging from 96.7% to 40.3% by tobacco smoke condensate (TSC). Cell viability of U373MG co-cultured with human brain microvascular endothelial cells (HBMECs) was 104.9% at the IC 50 value of TSC. Trans-epithelial electric resistance values drastically decreased 80% following 12-h incubation. The value was maintained until 48 h and then increased at 72-h incubation (85%). It then decreased to 75% at 120 h. Generation of ROS increased in a dose-dependent manner, ranging from 102.7% to 107.9%, when various concentrations of TSC (4-16 mg/mL) were administered to the U373MG monoculture. When TSC was added into U373MG co-cultured with HBMECs, production of ROS ranged from 101.7% to 102.6%, slightly increasing over 12 h. Maximum exposure-generated ROS of 104.8% was reached at 24 h. Cell cytotoxicity and oxidative stress levels in the U373MG co-culture model system with HBMECs were lower than U373MG monoculture. HBMECs effectively acted as a barrier to protect the astrocytes (U373MG) from toxicity of TSC.

  14. Contributions of 3D Cell Cultures for Cancer Research.

    Science.gov (United States)

    Ravi, Maddaly; Ramesh, Aarthi; Pattabhi, Aishwarya

    2017-10-01

    Cancer cell lines have contributed immensely in understanding the complex physiology of cancers. They are excellent material for studies as they offer homogenous samples without individual variations and can be utilised with ease and flexibility. Also, the number of assays and end-points one can study is almost limitless; with the advantage of improvising, modifying or altering several variables and methods. Literally, a new dimension to cancer research has been achieved by the advent of 3Dimensional (3D) cell culture techniques. This approach increased many folds the ways in which cancer cell lines can be utilised for understanding complex cancer biology. 3D cell culture techniques are now the preferred way of using cancer cell lines to bridge the gap between the 'absolute in vitro' and 'true in vivo'. The aspects of cancer biology that 3D cell culture systems have contributed include morphology, microenvironment, gene and protein expression, invasion/migration/metastasis, angiogenesis, tumour metabolism and drug discovery, testing chemotherapeutic agents, adaptive responses and cancer stem cells. We present here, a comprehensive review on the applications of 3D cell culture systems for these aspects of cancers. J. Cell. Physiol. 232: 2679-2697, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  15. Bidirectional apical-basal traffic of the cation-independent mannose-6-phosphate receptor in brain endothelial cells.

    Science.gov (United States)

    Siupka, Piotr; Hersom, Maria Ns; Lykke-Hartmann, Karin; Johnsen, Kasper B; Thomsen, Louiza B; Andresen, Thomas L; Moos, Torben; Abbott, N Joan; Brodin, Birger; Nielsen, Morten S

    2017-07-01

    Brain capillary endothelium mediates the exchange of nutrients between blood and brain parenchyma. This barrier function of the brain capillaries also limits passage of pharmaceuticals from blood to brain, which hinders treatment of several neurological disorders. Receptor-mediated transport has been suggested as a potential pharmaceutical delivery route across the brain endothelium, e.g. reports have shown that the transferrin receptor (TfR) facilitates transcytosis of TfR antibodies, but it is not known whether this recycling receptor itself traffics from apical to basal membrane in the process. Here, we elucidate the endosomal trafficking of the retrograde transported cation-independent mannose-6-phosphate receptor (MPR300) in primary cultures of brain endothelial cells (BECs) of porcine and bovine origin. Receptor expression and localisation of MPR300 in the endo-lysosomal system and trafficking of internalised receptor are analysed. We also demonstrate that MPR300 can undergo bidirectional apical-basal trafficking in primary BECs in co-culture with astrocytes. This is, to our knowledge, the first detailed study of retrograde transported receptor trafficking in BECs, and the study demonstrates that MPR300 can be transported from the luminal to abluminal membrane and reverse. Such trafficking of MPR300 suggests that retrograde transported receptors in general may provide a mechanism for transport of pharmaceuticals into the brain.

  16. Advances in culture and manipulation of human pluripotent stem cells.

    Science.gov (United States)

    Qian, X; Villa-Diaz, L G; Krebsbach, P H

    2013-11-01

    Recent advances in the understanding of pluripotent stem cell biology and emerging technologies to reprogram somatic cells to a stem cell-like state are helping bring stem cell therapies for a range of human disorders closer to clinical reality. Human pluripotent stem cells (hPSCs) have become a promising resource for regenerative medicine and research into early development because these cells are able to self-renew indefinitely and are capable of differentiation into specialized cell types of all 3 germ layers and trophoectoderm. Human PSCs include embryonic stem cells (hESCs) derived from the inner cell mass of blastocyst-stage embryos and induced pluripotent stem cells (hiPSCs) generated via the reprogramming of somatic cells by the overexpression of key transcription factors. The application of hiPSCs and the finding that somatic cells can be directly reprogrammed into different cell types will likely have a significant impact on regenerative medicine. However, a major limitation for successful therapeutic application of hPSCs and their derivatives is the potential xenogeneic contamination and instability of current culture conditions. This review summarizes recent advances in hPSC culture and methods to induce controlled lineage differentiation through regulation of cell-signaling pathways and manipulation of gene expression as well as new trends in direct reprogramming of somatic cells.

  17. Cell culture supernatants for detection perforin ELISA

    African Journals Online (AJOL)

    Najwa

    2014-02-19

    Feb 19, 2014 ... (2001). The lymphocytes were isolated from the peripheral heap- rinized whole blood as follows: 3 ml of blood was centrifuged at. 1000 rpm for 15 min, buffy coat was collected in a 10 ml centrifuge tubes and diluted with 5 ml RPMI 1640 (cell suspension), 5 ml of the diluted cell suspension was layered on 3 ...

  18. Growth and phenotypic characteristics of human nevus cells in culture.

    Science.gov (United States)

    Mancianti, M L; Herlyn, M; Weil, D; Jambrosic, J; Rodeck, U; Becker, D; Diamond, L; Clark, W H; Koprowski, H

    1988-02-01

    Nevus cells were isolated from the three cutaneous components, epidermis, basal layer, and dermis, of nonmalignant pigmented lesions and were cultured separately in the presence or absence of the phorbol ester 12-0-tetradecanoyl phorbol-13-acetate in medium that supports the rapid proliferation of melanocytic cells. The separation procedure used provided cultures that were essentially free from normal melanocytes (dermis) or fibroblasts (epidermis). In short term culture, nevus cells of all skin compartments expressed markers associated with differentiated melanocytes, such as presence of premelanosomes and melanosomes and elevated tyrosinase levels. Nevus cells also expressed melanoma-associated antigens, such as NGF-receptor, transferrin-related p97, proteoglycan, and HLA-DR as detected with monoclonal antibodies. After several subpassages, cells showed a decreased expression of melanoma-associated antigens, decreased tyrrosinase levels, and melanosomes could no longer be detected. Morphologically, these cells were similar to fibroblasts. The disappearance of melanoma-associated cell surface antigens was concomitant with the appearance of a melanocyte-associated 145 kd protein that might serve as a marker of fibroblast-like differentiation in nevus cells and normal melanocytes. Nevus cell cultures grown in the presence of 12-0-tetradecanoyl phorbol-13-acetate maintained a stable differentiated phenotype throughout their lifespan. As reported earlier, nevus cells in culture, irrespective of the presence or absence of 12-0-tetradecanoyl phorbol-13-acetate, have a finite lifespan in vitro, grow anchorage-independent in soft agar, but do not form tumors when xenografted to nude mice. These studies demonstrate that nevus cells isolated from the epidermal, basal layer, and dermal components of lesional skin can serve as models to characterize the initial steps of tumor progression in a human cell system.

  19. Single nucleotide variations in cultured cancer cells: Effect of mismatch repair.

    Science.gov (United States)

    Panyutin, Igor G; Panyutin, Irina V; Powell-Castilla, Ian; Felix, Laura; Neumann, Ronald D

    2017-10-01

    We assessed single nucleotide variations (SNVs) between individual cells in two cancer cell lines; DU145, from brain metastasis of prostate tumor with deficient mismatch repair; and HT1080, a fibrosarcoma cell line. Clones of individual cells were isolated, and sequenced using Ion Ampliseq comprehensive cancer panel that covered the exomes of 409 oncogenes and tumor suppressor genes. Five clones of DU145 and four clones of HT1080 cells were analyzed. We found from 7 to 12 unique SNVs between DU145 clones, while HT1080 clones showed no more than one unique SNV. We then sub-cloned individual cells from some of these isolated clones of DU145 and HT1080 cells. The sub-clones were expanded from a single cell to approximately one million cells after about 20 cell divisions. The sub-clones of DU145 cells had from one to four new unique SNVs within the sequenced regions. No unique SNVs were found between sub-clones of HT1080 cells. Our data demonstrate that the extent of genetic variation at the single nucleotide level in cultured cancer cells is significantly affected by the status of the DNA mismatch repair system. Published by Elsevier B.V.

  20. The fibrinolytic system facilitates tumor cell migration across the blood-brain barrier in experimental melanoma brain metastasis

    International Nuclear Information System (INIS)

    Perides, George; Zhuge, Yuzheng; Lin, Tina; Stins, Monique F; Bronson, Roderick T; Wu, Julian K

    2006-01-01

    Patients with metastatic tumors to the brain have a very poor prognosis. Increased metastatic potential has been associated with the fibrinolytic system. We investigated the role of the fibrinolytic enzyme plasmin in tumor cell migration across brain endothelial cells and growth of brain metastases in an experimental metastatic melanoma model. Metastatic tumors to the brain were established by direct injection into the striatum or by intracarotid injection of B16F10 mouse melanoma cells in C57Bl mice. The role of plasminogen in the ability of human melanoma cells to cross a human blood-brain barrier model was studied on a transwell system. Wild type mice treated with the plasmin inhibitor epsilon-aminocaproic acid (EACA) and plg -/- mice developed smaller tumors and survived longer than untreated wild type mice. Tumors metastasized to the brain of wild type mice treated with EACA and plg -/- less efficiently than in untreated wild type mice. No difference was observed in the tumor growth in any of the three groups of mice. Human melanoma cells were able to cross the human blood-brain barrier model in a plasmin dependent manner. Plasmin facilitates the development of tumor metastasis to the brain. Inhibition of the fibrinolytic system could be considered as means to prevent tumor metastasis to the brain

  1. Stability of resazurin in buffers and mammalian cell culture media

    DEFF Research Database (Denmark)

    Rasmussen, Eva; Nicolaisen, G.M.

    1999-01-01

    The utility of a ferricyanide/ferrocyanide system used in the AlamarBlue(TM) (Serotec, Oxford, UK) vital. dye to inhibit the reduction of resazurin by mammalian cell culture media is questioned. Resazurin was found to be relatively stable when dissolved in phosphate-buffered saline (PBS). The use...... of HEPES resulted in a huge immediate dye reduction, which was significantly enhanced by exposure to diffuse light from fluorescent tubes in the laboratory 8 h per day. The reduction of resazurin by various cell culture media was time and temperature dependent, and it was significantly enhanced......'s nutrient mixture F-10 and F-12. Fetal calf serum (5-20%) slightly decreased resazurin reduction during the first 2 days of incubation. The reduction of resazurin by mammalian cell culture media do not appear to be problematic under normal culture conditions, and it is primarily dependent upon the presence...

  2. Neuropeptide Mapping of Dimmed Cells of Adult Drosophila Brain

    Science.gov (United States)

    Diesner, Max; Predel, Reinhard; Neupert, Susanne

    2018-01-01

    Neuropeptides are structurally highly diverse messenger molecules that act as regulators of many physiological processes such as development, metabolism, reproduction or behavior in general. Differentiation of neuropeptidergic cells often corresponds with the presence of the transcription factor DIMMED. In the central nervous system of the fruit fly Drosophila melanogaster, DIMMED commonly occurs in neuroendocrine neurons that release peptides as neurohormones but also in interneurons with complex branching patterns. Fly strains with green fluorescence protein (GFP)-expressing dimmed cells make it possible to systematically analyze the processed neuropeptides in these cells. In this study, we mapped individual GFP-expressing neurons of adult D. melanogaster from the dimmed (c929)>GFP line. Using single cell mass spectrometry, we analyzed 10 types of dimmed neurons from the brain/gnathal ganglion. These cells included neuroendocrine cells with projection into the retrocerebral complex but also a number of large interneurons. Resulting mass spectra not only provided comprehensive data regarding mature products from 13 neuropeptide precursors but also evidence for the cellular co-localization of neuropeptides from different neuropeptide genes. The results can be implemented in a neuroanatomical map of the D. melanogaster brain. [Figure not available: see fulltext.

  3. Fabrication of a thermoresponsive cell culture dish: a key technology for cell sheet tissue engineering

    Directory of Open Access Journals (Sweden)

    Jun Kobayashi and Teruo Okano

    2010-01-01

    Full Text Available This article reviews the properties and characterization of an intelligent thermoresponsive surface, which is a key technology for cell sheet-based tissue engineering. Intelligent thermoresponsive surfaces grafted with poly(N-isopropylacrylamide exhibit hydrophilic/hydrophobic alteration in response to temperature change. Cultured cells are harvested on thermoresponsive cell culture dishes by decreasing the temperature without the use of digestive enzymes or chelating agents. Our group has developed cell sheet-based tissue engineering for therapeutic uses with single layer or multilayered cell sheets, which were recovered from the thermoresponsive cell culture dish. Using surface derivation techniques, we developed a new generation of thermoresponsive cell culture dishes to improve culture conditions. We also designed a new methodology for constructing well-defined organs using microfabrication techniques.

  4. Characterisation and germline transmission of cultured avian primordial germ cells.

    Science.gov (United States)

    Macdonald, Joni; Glover, James D; Taylor, Lorna; Sang, Helen M; McGrew, Michael J

    2010-11-29

    Avian primordial germ cells (PGCs) have significant potential to be used as a cell-based system for the study and preservation of avian germplasm, and the genetic modification of the avian genome. It was previously reported that PGCs from chicken embryos can be propagated in culture and contribute to the germ cell lineage of host birds. We confirm these results by demonstrating that PGCs from a different layer breed of chickens can be propagated for extended periods in vitro. We demonstrate that intracellular signalling through PI3K and MEK is necessary for PGC growth. We carried out an initial characterisation of these cells. We find that cultured PGCs contain large lipid vacuoles, are glycogen rich, and express the stem cell marker, SSEA-1. These cells also express the germ cell-specific proteins CVH and CDH. Unexpectedly, using RT-PCR we show that cultured PGCs express the pluripotency genes c-Myc, cKlf4, cPouV, cSox2, and cNanog. Finally, we demonstrate that the cultured PGCs will migrate to and colonise the forming gonad of host embryos. Male PGCs will colonise the female gonad and enter meiosis, but are lost from the gonad during sexual development. In male hosts, cultured PGCs form functional gametes as demonstrated by the generation of viable offspring. The establishment of in vitro cultures of germline competent avian PGCs offers a unique system for the study of early germ cell differentiation and also a comparative system for mammalian germ cell development. Primary PGC lines will form the basis of an alternative technique for the preservation of avian germplasm and will be a valuable tool for transgenic technology, with both research and industrial applications.

  5. Characterisation and germline transmission of cultured avian primordial germ cells.

    Directory of Open Access Journals (Sweden)

    Joni Macdonald

    Full Text Available BACKGROUND: Avian primordial germ cells (PGCs have significant potential to be used as a cell-based system for the study and preservation of avian germplasm, and the genetic modification of the avian genome. It was previously reported that PGCs from chicken embryos can be propagated in culture and contribute to the germ cell lineage of host birds. PRINCIPAL FINDINGS: We confirm these results by demonstrating that PGCs from a different layer breed of chickens can be propagated for extended periods in vitro. We demonstrate that intracellular signalling through PI3K and MEK is necessary for PGC growth. We carried out an initial characterisation of these cells. We find that cultured PGCs contain large lipid vacuoles, are glycogen rich, and express the stem cell marker, SSEA-1. These cells also express the germ cell-specific proteins CVH and CDH. Unexpectedly, using RT-PCR we show that cultured PGCs express the pluripotency genes c-Myc, cKlf4, cPouV, cSox2, and cNanog. Finally, we demonstrate that the cultured PGCs will migrate to and colonise the forming gonad of host embryos. Male PGCs will colonise the female gonad and enter meiosis, but are lost from the gonad during sexual development. In male hosts, cultured PGCs form functional gametes as demonstrated by the generation of viable offspring. CONCLUSIONS: The establishment of in vitro cultures of germline competent avian PGCs offers a unique system for the study of early germ cell differentiation and also a comparative system for mammalian germ cell development. Primary PGC lines will form the basis of an alternative technique for the preservation of avian germplasm and will be a valuable tool for transgenic technology, with both research and industrial applications.

  6. Integration of embryonic stem cells in metanephric kidney organ culture.

    Science.gov (United States)

    Steenhard, Brooke M; Isom, Kathryn S; Cazcarro, Patricia; Dunmore, Judy H; Godwin, Alan R; St John, Patricia L; Abrahamson, Dale R

    2005-06-01

    Many stages of nephrogenesis can be studied using cultured embryonic kidneys, but there is no efficient technique available to readily knockdown or overexpress transgenes for rapid evaluation of resulting phenotypes. Embryonic stem (ES) cells have unlimited developmental potential and can be manipulated at the molecular genetic level by a variety of methods. The aim of this study was to determine if ES cells could respond to developmental signals within the mouse embryonic day 12 to embryonic day 13 (E12 to E13) kidney microenvironment and incorporate into kidney structures. ROSA26 ES cells were shown to express beta-galactosidase ubiquitously when cultured in the presence of leukemia inhibitory factor to suppress differentiation. When these cells were microinjected into E12 to E13 metanephroi and then placed in transwell organ culture, ES cell-derived, beta-galactosidase-positive cells were identified in epithelial structures resembling tubules. On rare occasions, individual ES cells were observed in structures resembling glomerular tufts. Electron microscopy showed that the ES cell-derived tubules were surrounded by basement membrane and had apical microvilli and junctional complexes. Marker analysis revealed that a subset of these epithelial tubules bound Lotus tetragonolobus and expressed alpha(1) Na(+)/K(+) ATPase. ES cells were infected before injection with a cytomegalovirus promoter-green fluorescence protein (GFP) adenovirus and GFP expression was found as early as 18 h, persisting for up to 48 h in cultured kidneys. This ES cell technology may achieve the objective of obtaining a versatile cell culture system in which molecular interventions can be used in vitro and consequences of these perturbations on the normal kidney development program in vivo can be studied.

  7. Transcriptome analysis of primary bovine extra-embryonic cultured cells

    Directory of Open Access Journals (Sweden)

    Séverine A. Degrelle

    2015-12-01

    Full Text Available The dataset described in this article pertains to the article by Hue et al. (2015 entitled “Primary bovine extra-embryonic cultured cells: A new resource for the study of in vivo peri-implanting phenotypes and mesoderm formation” [1]. In mammals, extra-embryonic tissues are essential to support not only embryo patterning but also embryo survival, especially in late implanting species. These tissues are composed of three cell types: trophoblast (bTCs, endoderm (bXECs and mesoderm (bXMCs. Until now, it is unclear how these cells interact. In this study, we have established primary cell cultures of extra-embryonic tissues from bovine embryos collected at day-18 after artificial insemination. We used our homemade bovine 10K array (GPL7417 to analyze the gene expression profiles of these primary extra-embryonic cultured cells compared to the corresponding cells from in vivo micro-dissected embryos. Here, we described the experimental design, the isolation of bovine extra-embryonic cell types as well as the microarray expression analysis. The dataset has been deposited in Gene Expression Omnibus (GEO (accession number GSE52967. Finally, these primary cell cultures were a powerful tool to start studying their cellular properties, and will further allow in vitro studies on cellular interactions among extra-embryonic tissues, and potentially between extra-embryonic vs embryonic tissues.

  8. Animal-cell culture in aqueous two-phase systems

    NARCIS (Netherlands)

    Zijlstra, G.M.

    1998-01-01

    In current industrial biotechnology, animal-cell culture is an important source of therapeutic protein products. The conventional animal-cell production processes, however, include many unit operations as part of the fermentation and downstream processing strategy. The research described in

  9. Endothelial cell cultures as a tool in biomaterial research

    NARCIS (Netherlands)

    Kirkpatrick, CJ; Otto, M; van Kooten, T; Krump, [No Value; Kriegsmann, J; Bittinger, F

    1999-01-01

    Progress in biocompatibility and tissue engineering would today be inconceivable without the aid of in vitro techniques. Endothelial cell cultures represent a valuable tool not just in haemocompatibility testing, but also in the concept of designing hybrid organs. In the past endothelial cells (EC)

  10. Cell culture plastics with immobilized interleukin-4 for monocyte differentiation

    DEFF Research Database (Denmark)

    Hansen, Morten; Hjortø, Gertrud Malene; Met, Ozcan

    2011-01-01

    Standard cell culture plastic was surface modified by passive adsorption or covalent attachment of interleukin (IL)-4 and investigated for its ability to induce differentiation of human monocytes into mature dendritic cells, a process dose-dependently regulated by IL-4. Covalent attachment of IL-...

  11. Challenges of culturing human norovirus in three-dimensional organoid intestinal cell culture models.

    Directory of Open Access Journals (Sweden)

    Efstathia Papafragkou

    Full Text Available Human noroviruses are the most common cause of acute gastroenteritis worldwide. Recently, cell culture systems have been described using either human embryonic intestinal epithelial cells (Int-407 or human epithelial colorectal adenocarcinoma cells (Caco-2 growing on collagen-I porous micro carrier beads in a rotating bioreactor under conditions of physiological fluid shear. Here, we describe the efforts from two independent laboratories to implement this three dimensional (3D cell culture system for the replication of norovirus. Int-407 and Caco-2 were grown in a rotating bioreactor for up to 28 days. Prior to infection, cells were screened for the presence of microvilli by electron microscopy and stained for junction proteins (zonula occludens-1, claudin-1, and β-catenin. Differentiated 3D cells were transferred to 24-well plates and infected with bacteria-free filtrates of various norovirus genotypes (GI.1, GI.3, GI.8, GII.2, GII.4, GII.7, and GII.8. At 12 h, 24 h, and 48 h post inoculation, viral RNA from both cells and supernatants were collected and analyzed for norovirus RNA by real-time reverse transcription PCR. Despite observations of high expression of junction proteins and microvilli development in stained thin sections, our data suggest no significant increase in viral titer based on norovirus RNA copy number during the first 48 h after inoculation for the different samples and virus culture conditions tested. Our combined efforts demonstrate that 3D cell culture models using Int-407 or Caco-2 cells do not support norovirus replication and highlight the complexity and difficulty of developing a reproducible in vitro cell culture system for human norovirus.

  12. Time evolution of cell size distributions in dense cell cultures

    Science.gov (United States)

    Khain, Evgeniy

    2015-03-01

    Living cells in a dense system are all in contact with each other. The common assumption is that such cells stop dividing due to a lack of space. Recent experimental observations have shown, however, that cells continue dividing for a while, but other cells in the system must shrink, to allow the newborn cells to grow to a normal size. Due to these ``pressure'' effects, the average cell size dramatically decreases with time, and the dispersion in cell sizes decreases, too. The collective cell behavior becomes even more complex when the system is expanding: cells near the edges are larger and migrate faster, while cells deep inside the colony are smaller and move slower. This exciting experimental data still needs to be described theoretically, incorporating the distribution of cell sizes in the system. We propose a mathematical model for time evolution of cell size distribution both in a closed and open system. The model incorporates cell proliferation, cell growth after division, cell shrinking due to ``pressure'' from other cells, and possible cell detachment from the interface of a growing colony. This research sheds light on physical and biological mechanisms of cell response to a dense environment and on the role of mechanical stresses in determining the distribution of cell sizes in the system.

  13. Duchenne muscular dystrophy: normal ATP turnover in cultured cells

    International Nuclear Information System (INIS)

    Fox, I.H.; Bertorini, T.; Palmieri, G.M.A.; Shefner, R.

    1986-01-01

    This paper examines ATP metabolism in cultured muscle cells and fibroblasts from patients with Duchenne dystrophy. ATP and ADP levels were the same in cultured cells from normal subjects and patients and there was no difference in ATP synthesis or degradation. The ATP synthesis was measured by the incorporation of C 14-U-adenine into aTP and ADP. although there was a significant decrease in radioactively labelled ATP after incubation with deoxyglucose in Duchenne muscle cells, there was no difference in ATP concentration of ADP metabolism

  14. Cytopathogenicity of Naegleria for cultured neuroblastoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Fulford, D.E.

    1985-01-01

    The cytopathic activity of live Naegleria amoebae and cell-free lysates of Naegleria for B-103 rat neuroblastoma cells was investigated using a /sup 51/Cr release assay. Live amoebae and cell-free lysates of N. fowleri, N. australiensis, N. lovaniensis, and N. gruberi all induced sufficient damage to radiolabeled B-103 cells to cause a significant release of chromium. The cytotoxic activity present in the cell-free lysates of N. fowleri can be recovered in the supernatant fluid following centrifugation at 100,000xg and precipitation of the 100,000xg supernatant fluid with ammonium sulfate. Initial characterization of the cytotoxic factor indicates that it is a heat labile, pH sensitive, soluble protein. The cytotoxic activity is abolished by either extraction, unaffected by repeated freeze-thawing, and is not sensitive to inhibitors of proteolytic enzymes. Phospholipase A activity was detected in the cytotoxic ammonium sulfate precipitable material, suggesting that this enzyme activity may have a role in the cytotoxic activity of the cell-free lysates.

  15. Connecting Malfunctioning Glial Cells and Brain Degenerative Disorders.

    Science.gov (United States)

    Kaminsky, Natalie; Bihari, Ofer; Kanner, Sivan; Barzilai, Ari

    2016-06-01

    The DNA damage response (DDR) is a complex biological system activated by different types of DNA damage. Mutations in certain components of the DDR machinery can lead to genomic instability disorders that culminate in tissue degeneration, premature aging, and various types of cancers. Intriguingly, malfunctioning DDR plays a role in the etiology of late onset brain degenerative disorders such as Parkinson's, Alzheimer's, and Huntington's diseases. For many years, brain degenerative disorders were thought to result from aberrant neural death. Here we discuss the evidence that supports our novel hypothesis that brain degenerative diseases involve dysfunction of glial cells (astrocytes, microglia, and oligodendrocytes). Impairment in the functionality of glial cells results in pathological neuro-glial interactions that, in turn, generate a "hostile" environment that impairs the functionality of neuronal cells. These events can lead to systematic neural demise on a scale that appears to be proportional to the severity of the neurological deficit. Copyright © 2016 The Authors. Production and hosting by Elsevier Ltd.. All rights reserved.

  16. Connecting Malfunctioning Glial Cells and Brain Degenerative Disorders

    Directory of Open Access Journals (Sweden)

    Natalie Kaminsky

    2016-06-01

    Full Text Available The DNA damage response (DDR is a complex biological system activated by different types of DNA damage. Mutations in certain components of the DDR machinery can lead to genomic instability disorders that culminate in tissue degeneration, premature aging, and various types of cancers. Intriguingly, malfunctioning DDR plays a role in the etiology of late onset brain degenerative disorders such as Parkinson’s, Alzheimer’s, and Huntington’s diseases. For many years, brain degenerative disorders were thought to result from aberrant neural death. Here we discuss the evidence that supports our novel hypothesis that brain degenerative diseases involve dysfunction of glial cells (astrocytes, microglia, and oligodendrocytes. Impairment in the functionality of glial cells results in pathological neuro-glial interactions that, in turn, generate a “hostile” environment that impairs the functionality of neuronal cells. These events can lead to systematic neural demise on a scale that appears to be proportional to the severity of the neurological deficit.

  17. Amburana cearensis seed extract protects brain mitochondria from oxidative stress and cerebellar cells from excitotoxicity induced by glutamate.

    Science.gov (United States)

    Lima Pereira, Érica Patrícia; Santos Souza, Cleide; Amparo, Jessika; Short Ferreira, Rafael; Nuñez-Figueredo, Yanier; Gonzaga Fernandez, Luzimar; Ribeiro, Paulo Roberto; Braga-de-Souza, Suzana; Amaral da Silva, Victor Diogenes; Lima Costa, Silvia

    2017-09-14

    Amburana cearensis (Allemao) A.C.Sm. is a medicinal plant of the Brazilian Caatinga reported to present antioxidant and anti-inflammatory activity. This study aimed to evaluate the neuroprotective effect of the extracts obtained from the seeds of A. cearensis in primary cultures of cerebellar cells subjected to excitotoxicity induced by glutamate and brain mitochondria submitted to oxidative stress. and methods: Primary cultures of cerebellar cells were treated with the ethanol (ETAC), hexane (EHAC), dichloromethane (EDAC) and ethyl acetate (EAAC) extracts of the seeds of A.cearensis and subjected to excitotoxicity induced by glutamate (10µM). Mitochondria isolated from rat brains were submitted to oxidative stress and treated with ETAC. Only the EHAC extract reduced cell viability by 30% after 72h of treatment. Morphological analyses by Immunofluorescence showed positive staining for glutamine synthetase, β-III tubulin, GFAP and IBA1 similar to control cultures, indicating a better preservation of astrocytes, neurons and microglia, after excitotoxic damage induced by glutamate in cerebellar cultures treated with the extracts. The ETAC extract also protected mitochondria isolated from rat brains from oxidative stress, reducing the swelling, dissipation of the membrane potential, ROS production and calcium influx. Thus, this study suggests that the seed extracts from A. Cearensis exhibit neuroprotective potential against oxidative stress and excitotoxicity induced by glutamate and can be considered a potential therapeutic agent in the treatment of neurodegenerative diseases. Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.

  18. Establishing a stem cell culture laboratory for clinical trials

    Science.gov (United States)

    Sekiya, Elíseo Joji; Forte, Andresa; Kühn, Telma Ingrid Borges de Bellis; Janz, Felipe; Bydlowski, Sérgio Paulo; Alves, Adelson

    2012-01-01

    Adult stem/progenitor cells are found in different human tissues. An in vitro cell culture is needed for their isolation or for their expansion when they are not available in a sufficient quantity to regenerate damaged organs and tissues. The level of complexity of these new technologies requires adequate facilities, qualified personnel with experience in cell culture techniques, assessment of quality and clear protocols for cell production. The rules for the implementation of cell therapy centers involve national and international standards of good manufacturing practices. However, such standards are not uniform, reflecting the diversity of technical and scientific development. Here standards from the United States, the European Union and Brazil are analyzed. Moreover, practical solutions encountered for the implementation of a cell therapy center appropriate for the preparation and supply of cultured cells for clinical studies are described. Development stages involved the planning and preparation of the project, the construction of the facility, standardization of laboratory procedures and development of systems to prevent cross contamination. Combining the theoretical knowledge of research centers involved in the study of cells with the practical experience of blood therapy services that manage structures for cell transplantation is presented as the best potential for synergy to meet the demands to implement cell therapy centers. PMID:23049427

  19. Biophysical characteristics of cells cultured on cholesteryl ester liquid crystals.

    Science.gov (United States)

    Soon, Chin Fhong; Omar, Wan Ibtisam Wan; Berends, Rebecca F; Nayan, Nafarizal; Basri, Hatijah; Tee, Kian Sek; Youseffi, Mansour; Blagden, Nick; Denyer, Morgan Clive Thomas

    2014-01-01

    This study aimed at examining the biophysical characteristics of human derived keratinocytes (HaCaT) cultured on cholesteryl ester liquid crystals (CELC). CELC was previously shown to improve sensitivity in sensing cell contractions. Characteristics of the cell integrin expressions and presence of extracellular matrix (ECM) proteins on the liquid crystals were interrogated using various immunocytochemical techniques. The investigation was followed by characterization of the chemical properties of the liquid crystals (LC) after immersion in cell culture media using Fourier transform infrared spectroscopy (FTIR). The surface morphology of cells adhered to the LC was studied using atomic force microscopy (AFM). Consistent with the expressions of the integrins α2, α3 and β1, extracellular matrix proteins (laminin, collagen type IV and fibronectin) were found secreted by the HaCaT onto CELC and these proteins were also secreted by cells cultured on the glass substrates. FTIR analysis of the LC revealed the existence of spectrum assigned to cholesterol and ester moieties that are essential compounds for the metabolizing activities of keratinocytes. The immunostainings indicated that cell adhesion on the LC is mediated by self-secreted ECM proteins. As revealed by the AFM imaging, the constraint in cell membrane spread on the LC leads to the increase in cell surface roughness and thickness of cell membrane. The biophysical expressions of cells on biocompatible CELC suggested that CELC could be a new class of biological relevant material. Copyright © 2013 Elsevier Ltd. All rights reserved.

  20. Microfluidic bioreactors for culture of non-adherent cells

    DEFF Research Database (Denmark)

    Shah, Pranjul Jaykumar; Vedarethinam, Indumathi; Kwasny, Dorota

    2011-01-01

    Microfluidic bioreactors (μBR) are becoming increasingly popular for cell culture, sample preparation and analysis in case of routine genetic and clinical diagnostics. We present a novel μBR for non-adherent cells designed to mimic in vivo perfusion of cells based on diffusion of media through...... a sandwiched membrane. The culture chamber and perfusion chamber are separated by a sandwiched membrane and each chamber has separate inlet/outlets for easy loading/unloading of cells and perfusion of the media. The perfusion of media and exchange of nutrients occur through the sandwiched membrane, which...... was also verified with simulations. Finally, we present the application of this device for cytogenetic sample preparation, whereby we culture and arrest peripheral T-lymphocytes in metaphase and later fix them in the μBR. The expansion of T-lymphocytes from an unknown patient sample was quantified by means...

  1. Hydrodynamic effects on cells in agitated tissue culture reactors

    Science.gov (United States)

    Cherry, R. S.; Papoutsakis, E. T.

    1986-01-01

    The mechanisms by which hydrodynamic forces can affect cells grown on microcarrier beads in agitated cell culture reactors were investigated by analyzing the motion of microcarriers relative to the surrounding fluid, to each other, and to moving or stationary solid surfaces. It was found that harmful effects on cell cultures that have been previously attributed to shear can be better explained as the effects of turbulence (of a size scale comparable to the microcarriers or the spacing between them) or collisions. The primary mechanisms of cell damage involve direct interaction between microcarriers and turbulent eddies, collisions between microcarriers in turbulent flow, and collisions against the impeller or other solid surfaces. The implications of these analytical results for the design of tissue culture reactors are discussed.

  2. Miniature Bioreactor System for Long-Term Cell Culture

    Science.gov (United States)

    Gonda, Steve R.; Kleis, Stanley J.; Geffert, Sandara K.

    2010-01-01

    A prototype miniature bioreactor system is designed to serve as a laboratory benchtop cell-culturing system that minimizes the need for relatively expensive equipment and reagents and can be operated under computer control, thereby reducing the time and effort required of human investigators and reducing uncertainty in results. The system includes a bioreactor, a fluid-handling subsystem, a chamber wherein the bioreactor is maintained in a controlled atmosphere at a controlled temperature, and associated control subsystems. The system can be used to culture both anchorage-dependent and suspension cells, which can be either prokaryotic or eukaryotic. Cells can be cultured for extended periods of time in this system, and samples of cells can be extracted and analyzed at specified intervals. By integrating this system with one or more microanalytical instrument(s), one can construct a complete automated analytical system that can be tailored to perform one or more of a large variety of assays.

  3. A novel closed cell culture device for fabrication of corneal epithelial cell sheets.

    Science.gov (United States)

    Nakajima, Ryota; Kobayashi, Toyoshige; Moriya, Noboru; Mizutani, Manabu; Kan, Kazutoshi; Nozaki, Takayuki; Saitoh, Kazuo; Yamato, Masayuki; Okano, Teruo; Takeda, Shizu

    2015-11-01

    Automation technology for cell sheet-based tissue engineering would need to optimize the cell sheet fabrication process, stabilize cell sheet quality and reduce biological contamination risks. Biological contamination must be avoided in clinical settings. A closed culture system provides a solution for this. In the present study, we developed a closed culture device called a cell cartridge, to be used in a closed cell culture system for fabricating corneal epithelial cell sheets. Rabbit limbal epithelial cells were cultured on the surface of a porous membrane with 3T3 feeder cells, which are separate from the epithelial cells in the cell cartridges and in the cell-culture inserts as a control. To fabricate the stratified cell sheets, five different thicknesses of the membranes which were welded to the cell cartridge, were examined. Multilayered corneal epithelial cell sheets were fabricated in cell cartridges that were welded to a 25 µm-thick gas-permeable membrane, which was similar to the results with the cell-culture inserts. However, stratification of corneal epithelial cell sheets did not occur with cell cartridges that were welded to 100-300 µm-thick gas-permeable membranes. The fabricated cell sheets were evaluated by histological analyses to examine the expression of corneal epithelial-specific markers. Immunohistochemical analyses showed that a putative stem cell marker, p63, a corneal epithelial differentiation maker, CK3, and a barrier function marker, Claudin-1, were expressed in the appropriate position in the cell sheets. These results suggest that the cell cartridge is effective for fabricating corneal epithelial cell sheets. Copyright © 2012 John Wiley & Sons, Ltd.

  4. A cross-cultural study on impaired self-awareness in Japanese patients with brain dysfunction.

    Science.gov (United States)

    Prigatano, G P; Ogano, M; Amakusa, B

    1997-04-01

    Japanese patients with brain dysfunction (21 with severe traumatic brain injury [TBI], 21 with right, and 21 with left cerebral vascular accidents [CVA]) were asked to make behavioral ratings regarding their competencies in several areas. Relatives of patients and physical therapists who treated them also rated each patient's behavioral competency. Japanese patients with TBI overestimated their behavioral competencies compared with therapists ratings, but not relatives' ratings. Japanese patients with TBI overstimated self-care skills but not their ability to interact in socioemotional situations. Patients who had right and left CVA did not differ in their mean ratings of behavioral competency. Among all patient groups, there was no correlation between self-reported competencies and performance on a neuropsychological test. Family ratings of patients' behavioral competencies correlated with the patients' neuropsychological test performance. Post hoc analyses of patients with TBI suggest that speed of finger tapping related to an impaired self-awareness. Whereas cultural factors may influence self-reports of behavioral competency, patients across cultures with brain dysfunction seem to have reduced insight into their actual level of neuropsychological functioning.

  5. The replacement of serum by hormones in cell culture media.

    Science.gov (United States)

    Sato, G; Hayashi, I

    1976-12-01

    The replacement of serum by hormones in cell culture media. (Reemplazo del suero por hormonas en el medio de cultivo de células). Arch. Biol. Med. Exper. 10: 120-121, 1976. The serum used in cell culture media can be replaced by a mixture of hormones and some accesory blood factors. The pituitary cell line GH3 can be grown in a medium in which serum is replaced by triiodothyronine, transferrin, parathormone, tyrotrophin releasing hormone and somatomedins. Hela and BHK cell strains can also be grown in serum free medium supplemented with hormones. Each cell type appears to have different hormonal requirements yet it may found that some hormones are required for most cell types.

  6. Therapeutic touch stimulates the proliferation of human cells in culture.

    Science.gov (United States)

    Gronowicz, Gloria A; Jhaveri, Ankur; Clarke, Libbe W; Aronow, Michael S; Smith, Theresa H

    2008-04-01

    Our objective was to assess the effect of Therapeutic Touch (TT) on the proliferation of normal human cells in culture compared to sham and no treatment. Several proliferation techniques were used to confirm the results, and the effect of multiple 10-minute TT treatments was studied. Fibroblasts, tendon cells (tenocytes), and bone cells (osteoblasts) were treated with TT, sham, or untreated for 2 weeks, and then assessed for [(3)H]-thymidine incorporation into the DNA, and immunocytochemical staining for proliferating cell nuclear antigen (PCNA). The number of PCNA-stained cells was also quantified. For 1 and 2 weeks, varying numbers of 10-minute TT treatments were administered to each cell type to determine whether there was a dose-dependent effect. TT administered twice a week for 2 weeks significantly stimulated proliferation of fibroblasts, tenocytes, and osteoblasts in culture (p = 0.04, 0.01, and 0.01, respectively) compared to untreated control. These data were confirmed by PCNA immunocytochemistry. In the same experiments, sham healer treatment was not significantly different from the untreated cultures in any group, and was significantly less than TT treatment in fibroblast and tenocyte cultures. In 1-week studies involving the administration of multiple 10-minute TT treatments, four and five applications significantly increased [(3)H]-thymidine incorporation in fibroblasts and tenocytes, respectively, but not in osteoblasts. With different doses of TT for 2 weeks, two 10-minute TT treatments per week significantly stimulated proliferation in all cell types. Osteoblasts also responded to four treatments per week with a significant increase in proliferation. Additional TT treatments (five per week for 2 weeks) were not effective in eliciting increased proliferation compared to control in any cell type. A specific pattern of TT treatment produced a significant increase in proliferation of fibro-blasts, osteoblasts, and tenocytes in culture. Therefore, TT may

  7. Hypoxic contraction of cultured pulmonary vascular smooth muscle cells

    International Nuclear Information System (INIS)

    Murray, T.R.; Chen, L.; Marshall, B.E.; Macarak, E.J.

    1990-01-01

    The cellular events involved in generating the hypoxic pulmonary vasoconstriction response are not clearly understood, in part because of the multitude of factors that alter pulmonary vascular tone. The goal of the present studies was to determine if a cell culture preparation containing vascular smooth muscle (VSM) cells could be made to contract when exposed to a hypoxic atmosphere. Cultures containing only fetal bovine pulmonary artery VSM cells were assessed for contractile responses to hypoxic stimuli by two methods. In the first, tension forces generated by cells grown on a flexible growth surface (polymerized polydimethyl siloxane) were manifested as wrinkles and distortions of the surface under the cells. Wrinkling of the surface was noted to progressively increase with time as the culture medium bathing the cells was made hypoxic (PO2 approximately 25 mmHg). The changes were sometimes reversible upon return to normoxic conditions and appeared to be enhanced in cells already exhibiting evidence of some baseline tone. Repeated passage in culture did not diminish the hypoxic response. Evidence for contractile responses to hypoxia was also obtained from measurements of myosin light chain (MLC) phosphorylation. Conversion of MLC to the phosphorylated species is an early step in the activation of smooth muscle contraction. Lowering the PO2 in the culture medium to 59 mmHg caused a 45% increase in the proportion of MLC in the phosphorylated form as determined by two-dimensional gel electrophoresis. Similarly, cultures preincubated for 4 h with 32P and then exposed to normoxia or hypoxia for a 5-min experimental period showed more than twice as much of the label in MLCs of the hypoxic cells

  8. Animal-cell culture media: History, characteristics, and current issues.

    Science.gov (United States)

    Yao, Tatsuma; Asayama, Yuta

    2017-04-01

    Cell culture technology has spread prolifically within a century, a variety of culture media has been designed. This review goes through the history, characteristics and current issues of animal-cell culture media. A literature search was performed on PubMed and Google Scholar between 1880 and May 2016 using appropriate keywords. At the dawn of cell culture technology, the major components of media were naturally derived products such as serum. The field then gradually shifted to the use of chemical-based synthetic media because naturally derived ingredients have their disadvantages such as large batch-to-batch variation. Today, industrially important cells can be cultured in synthetic media. Nevertheless, the combinations and concentrations of the components in these media remain to be optimized. In addition, serum-containing media are still in general use in the field of basic research. In the fields of assisted reproductive technologies and regenerative medicine, some of the medium components are naturally derived in nearly all instances. Further improvements of culture media are desirable, which will certainly contribute to a reduction in the experimental variation, enhance productivity among biopharmaceuticals, improve treatment outcomes of assisted reproductive technologies, and facilitate implementation and popularization of regenerative medicine.

  9. Dose verification by OSLDs in the irradiation of cell cultures

    International Nuclear Information System (INIS)

    Meca C, E. A.; Bourel, V.; Notcovich, C.; Duran, H.

    2015-10-01

    The determination of value of irradiation dose presents difficulties when targets are irradiated located in regions where electronic equilibrium of charged particle is not reached, as in the case of irradiation -in vitro- of cell lines monolayer-cultured, in culture dishes or flasks covered with culture medium. The present study aimed to implement a methodology for dose verification in irradiation of cells in culture media by optically stimulated luminescence dosimetry (OSLD). For the determination of the absorbed dose in terms of cell proliferation OSL dosimeters of aluminum oxide doped with carbon (Al 2 O 3 :C) were used, which were calibrated to the irradiation conditions of culture medium and at doses that ranged from 0.1 to 15 Gy obtained with a linear accelerator of 6 MV photons. Intercomparison measurements were performed with an ionization chamber of 6 cm 3 . Different geometries were evaluated by varying the thicknesses of solid water, air and cell culture medium. The results showed deviations below 2.2% when compared with the obtained doses of OSLDs and planning system used. Also deviations were observed below 3.4% by eccentric points of the irradiation plane, finding homogeneous dose distribution. Uncertainty in the readings was less than 2%. The proposed methodology contributes a contribution in the dose verification in this type of irradiations, eliminating from the calculation uncertainties, potential errors in settling irradiation or possible equipment failure with which is radiating. It also provides certainty about the survival curves to be plotted with the experimental data. (Author)

  10. Nonlinear Dynamic Theory of Acute Cell Injuries and Brain Ischemia

    Science.gov (United States)

    Taha, Doaa; Anggraini, Fika; Degracia, Donald; Huang, Zhi-Feng

    2015-03-01

    Cerebral ischemia in the form of stroke and cardiac arrest brain damage affect over 1 million people per year in the USA alone. In spite of close to 200 clinical trials and decades of research, there are no treatments to stop post-ischemic neuron death. We have argued that a major weakness of current brain ischemia research is lack of a deductive theoretical framework of acute cell injury to guide empirical studies. A previously published autonomous model based on the concept of nonlinear dynamic network was shown to capture important facets of cell injury, linking the concept of therapeutic to bistable dynamics. Here we present an improved, non-autonomous formulation of the nonlinear dynamic model of cell injury that allows multiple acute injuries over time, thereby allowing simulations of both therapeutic treatment and preconditioning. Our results are connected to the experimental data of gene expression and proteomics of neuron cells. Importantly, this new model may be construed as a novel approach to pharmacodynamics of acute cell injury. The model makes explicit that any pro-survival therapy is always a form of sub-lethal injury. This insight is expected to widely influence treatment of acute injury conditions that have defied successful treatment to date. This work is supported by NIH NINDS (NS081347) and Wayne State University President's Research Enhancement Award.

  11. Exposure to lipopolysaccharide and/or unconjugated bilirubin impair the integrity and function of brain microvascular endothelial cells.

    Directory of Open Access Journals (Sweden)

    Filipa L Cardoso

    Full Text Available BACKGROUND: Sepsis and jaundice are common conditions in newborns that can lead to brain damage. Though lipopolysaccharide (LPS is known to alter the integrity of the blood-brain barrier (BBB, little is known on the effects of unconjugated bilirubin (UCB and even less on the joint effects of UCB and LPS on brain microvascular endothelial cells (BMEC. METHODOLOGY/PRINCIPAL FINDINGS: Monolayers of primary rat BMEC were treated with 1 µg/ml LPS and/or 50 µM UCB, in the presence of 100 µM human serum albumin, for 4 or 24 h. Co-cultures of BMEC with astroglial cells, a more complex BBB model, were used in selected experiments. LPS led to apoptosis and UCB induced both apoptotic and necrotic-like cell death. LPS and UCB led to inhibition of P-glycoprotein and activation of matrix metalloproteinases-2 and -9 in mono-cultures. Transmission electron microscopy evidenced apoptotic bodies, as well as damaged mitochondria and rough endoplasmic reticulum in BMEC by either insult. Shorter cell contacts and increased caveolae-like invaginations were noticeable in LPS-treated cells and loss of intercellular junctions was observed upon treatment with UCB. Both compounds triggered impairment of endothelial permeability and transendothelial electrical resistance both in mono- and co-cultures. The functional changes were confirmed by alterations in immunostaining for junctional proteins β-catenin, ZO-1 and claudin-5. Enlargement of intercellular spaces, and redistribution of junctional proteins were found in BMEC after exposure to LPS and UCB. CONCLUSIONS: LPS and/or UCB exert direct toxic effects on BMEC, with distinct temporal profiles and mechanisms of action. Therefore, the impairment of brain endothelial integrity upon exposure to these neurotoxins may favor their access to the brain, thus increasing the risk of injury and requiring adequate clinical management of sepsis and jaundice in the neonatal period.

  12. Isolation and Culture of Postnatal Stem Cells from Deciduous Teeth

    OpenAIRE

    Olávez, Daniela; Facultad de Odontología Universidad de Los Andes; Salmen, Siham; Instituto de Inmunología Clínica, Universidad de Los Andes.; Padrón, Karla; Facultad de Odontología. Univerisdad de Los Andes.; Lobo, Carmine; Facultad de Odontología. Univerisdad de Los Andes.; Díaz, Nancy; Facultad de Odontología, Universidad de Los Andes.; Berrueta, Lisbeth; Doctora en Inmunología por Instituto Venezolano de Investigaciones Científicas (IVIC). Instituto de Inmunología Clínica, Facultad de Medicina, Universidad de Los Andes, Venezuela.; Solorzanio, Eduvigis; Facultad de Odontología, Universidad de Los Andes.

    2014-01-01

    Background: Currently, degenerative diseases represent a public health problem; therefore, the development and implementation of strategies to fully or partially recover of damaged tissues has a special interest in the biomedical field. Therapeutic strategies based on mesenchymal stem cells transplantation from dental pulp have been proposed as an alternative. Purpose: To develop a mesenchymal stem cells culture isolated from dental pulp of deciduous teeth. Methods: The mesenchymal stem cells...

  13. Pluronic polyols in human lymphocyte cell line cultures.

    Science.gov (United States)

    Mizrahi, A

    1975-01-01

    Pluronic polyols markedly improved the growth of two human lymphocyte cell lines when added to the growth medium in concentrations of 0.05 to 0.1%. The results of the current studies suggest that, in addition to the protective effect of polyols against mechanical damage of mammalian cells in submerged cultures, the pluronic compounds may also, by lowering surface tension, facilitate transport of metabolites into cells and thus increase the growth rate. PMID:1063740

  14. Formation and action of oxygen activated species in cell cultures

    International Nuclear Information System (INIS)

    Hoffmann, M.E.; Meneghini, R.

    1982-01-01

    The differences of hydrogen peroxide sensibility of mammal cell lineages (man, mouse, chinese hamster) in culture are studied. The cellular survival and the frequency of DNA induced breaks by hydrogen peroxide are analysed. The efficiency of elimination of DNA breaks by cells is determined. The possible relation between the cell capacity of repair and its survival to hydrogen peroxide action is also discussed. (M.A.) [pt

  15. A microwell cell culture platform for the aggregation of pancreatic β-cells.

    Science.gov (United States)

    Bernard, Abigail B; Lin, Chien-Chi; Anseth, Kristi S

    2012-08-01

    Cell-cell contact between pancreatic β-cells is important for maintaining survival and normal insulin secretion. Various techniques have been developed to promote cell-cell contact between β-cells, but a simple yet robust method that affords precise control over three-dimensional (3D) β-cell cluster size has not been demonstrated. To address this need, we developed a poly(ethylene glycol) (PEG) hydrogel microwell platform using photolithography. This microwell cell-culture platform promotes the formation of 3D β-cell aggregates of defined sizes from 25 to 210 μm in diameter. Using this platform, mouse insulinoma 6 (MIN6) β-cells formed aggregates with cell-cell adherin junctions. These naturally formed cell aggregates with controllable sizes can be removed from the microwells for macroencapsulation, implantation, or other biological assays. When removed and subsequently encapsulated in PEG hydrogels, the aggregated cell clusters demonstrated improved cellular viability (>90%) over 7 days in culture, while the β-cells encapsulated as single cells maintained only 20% viability. Aggregated MIN6 cells also exhibited more than fourfold higher insulin secretion in response to a glucose challenge compared with encapsulated single β-cells. Further, the cell aggregates stained positively for E-cadherin, indicative of the formation of cell junctions. Using this hydrogel microwell cell-culture method, viable and functional β-cell aggregates of specific sizes were created, providing a platform from which other biologically relevant questions may be answered.

  16. HUMAN CELLS IN CULTURE: REVISlTED*

    African Journals Online (AJOL)

    advantages, e.g. the generation time is reduced to about. 1/10000 that of the ... or less reflects the cellular biology of the donor tissut:'Y .... X-linked. Autosomal recessive. Autosomal recessive. Autosomal recessive mothers of affected males, however, show that only 50% of the cell population is defective, which furnishes an.

  17. Brain morphology in children with nevoid basal cell carcinoma syndrome.

    Science.gov (United States)

    Shiohama, Tadashi; Fujii, Katsunori; Miyashita, Toshiyuki; Mizuochi, Hiromi; Uchikawa, Hideki; Shimojo, Naoki

    2017-04-01

    Brain morphology is tightly regulated by diverse signaling pathways. Hedgehog signaling is a candidate pathway considered responsible for regulating brain morphology. Nevoid basal cell carcinoma syndrome (NBCCS), caused by a PTCH1 mutation in the hedgehog signaling pathway, occasionally exhibits macrocephaly and medulloblastoma. Although cerebellar enlargement occurs in ptch1 heterozygous-deficient mice, its impact on human brain development remains unknown. We investigated the brain morphological characteristics of children with NBCCS. We evaluated brain T1-weighted images from nine children with NBCCS and 15 age-matched normal control (NC) children (mean [standard deviation], 12.2 [2.8] vs. 11.6 [2.3] years old). The diameters of the cerebrum, corpus callosum, and brain stem and the cerebellar volume were compared using two-tailed t-tests with Welch's correction. The transverse diameters (150.4 [9.9] vs. 136.0 [5.5] mm, P = 0.002) and longitudinal diameters (165.4 [8.0] vs. 151.3 [8.7] mm, P = 0.0007) of the cerebrum, cross-sectional area of the cerebellar vermis (18.7 [2.6] vs. 11.8 [1.7] cm 2 , P = 0.0001), and total volume of the cerebellar hemispheres (185.1 [13.0] vs. 131.9 [10.4] cm 3 , P = 0.0001) were significantly larger in the children with NBCCS than in NC children. Thinning of the corpus callosum and ventricular enlargement were also confirmed in children with NBCCS. We demonstrate that, on examination of the brain morphology, an increase in the size of the cerebrum, cerebellum, and cerebral ventricles is revealed in children with NBCCS compared to NC children. This suggests that constitutively active hedgehog signaling affects human brain morphology and the PI3K/AKT and RAS/MAPK pathways. © 2017 Wiley Periodicals, Inc.

  18. Decreased STAT3 Phosphorylation Mediates Cell Swelling in Ammonia-Treated Astrocyte Cultures

    Directory of Open Access Journals (Sweden)

    Arumugam R. Jayakumar

    2016-12-01

    Full Text Available Brain edema, due largely to astrocyte swelling, and the subsequent increase in intracranial pressure and brain herniation, are major complications of acute liver failure (ALF. Elevated level of brain ammonia has been strongly implicated in the development of astrocyte swelling associated with ALF. The means by which ammonia brings about astrocyte swelling, however, is incompletely understood. Recently, oxidative/nitrosative stress and associated signaling events, including activation of mitogen-activated protein kinases (MAPKs, as well as activation of the transcription factor, nuclear factor-kappaB (NF-κB, have been implicated in the mechanism of ammonia-induced astrocyte swelling. Since these signaling events are known to be regulated by the transcription factor, signal transducer and activator of transcription 3 (STAT3, we examined the state of STAT3 activation in ammonia-treated cultured astrocytes, and determined whether altered STAT3 activation and/or protein expression contribute to the ammonia-induced astrocyte swelling. STAT3 was found to be dephosphorylated (inactivated at Tyrosine705 in ammonia-treated cultured astrocytes. Total STAT3 protein level was also reduced in ammonia-treated astrocytes. We also found a significant increase in protein tyrosine phosphatase receptor type-1 (PTPRT-1 protein expression in ammonia-treated cultured astrocytes, and that inhibition of PTPRT-1 enhanced the phosphorylation of STAT3 after ammonia treatment. Additionally, exposure of cultured astrocytes to inhibitors of protein tyrosine phosphatases diminished the ammonia-induced cell swelling, while cultured astrocytes over-expressing STAT3 showed a reduction in the astrocyte swelling induced by ammonia. Collectively, these studies strongly suggest that inactivation of STAT3 represents a critical event in the mechanism of the astrocyte swelling associated with acute liver failure.

  19. Brain-computer-interfaces in their ethical, social and cultural contexts

    CERN Document Server

    Grübler, Gerd

    2014-01-01

    This volume summarizes the ethical, social and cultural contexts of interfacing brains and computers. It is intended for the interdisciplinary community of BCI stakeholders. Insofar, engineers, neuroscientists, psychologists, physicians, care-givers and also users and their relatives are concerned. For about the last twenty years brain-computer-interfaces (BCIs) have been investigated with increasing intensity and have in principle shown their potential to be useful tools in diagnostics, rehabilitation and assistive technology. The central promise of BCI technology is enabling severely impaired people in mobility, grasping, communication, and entertainment. Successful applications are for instance communication devices enabling locked-in patients in staying in contact with their environment, or prostheses enabling paralysed people in reaching and grasping. In addition to this, it serves as an introduction to the whole field of BCI for any interested reader.

  20. Altered eicosanoid production and phospholipid remodeling during cell culture.

    Science.gov (United States)

    Okuno, Toshiaki; Gijón, Miguel A; Zarini, Simona; Martin, Sarah A; Barkley, Robert M; Johnson, Christopher A; Ohba, Mai; Yokomizo, Takehiko; Murphy, Robert C

    2018-03-01

    The remodeling of PUFAs by the Lands cycle is responsible for the diversity of phospholipid molecular species found in cells. There have not been detailed studies of the alteration of phospholipid molecular species as a result of serum starvation or depletion of PUFAs that typically occurs during tissue culture. The time-dependent effect of cell culture on phospholipid molecular species in RAW 264.7 cells cultured for 24, 48, or 72 h was examined by lipidomic strategies. These cells were then stimulated to produce arachidonate metabolites derived from the cyclooxygenase pathway, thromboxane B 2 , PGE 2 , and PGD 2 , and the 5-lipoxygenase pathway, leukotriene (LT)B 4 , LTC 4 , and 5-HETE, which decreased with increasing time in culture. However, the 5-lipoxygenase metabolites of a 20:3 fatty acid, LTB 3 , all trans -LTB 3 , LTC 3 , and 5-hydroxyeicosatrienoic acid, time-dependently increased. Molecular species of arachidonate containing phospholipids were drastically remodeled during cell culture, with a new 20:3 acyl group being populated into phospholipids to replace increasingly scarce arachidonate. In addition, the amount of TNFα induced by lipopolysaccharide stimulation was significantly increased in the cells cultured for 72 h compared with 24 h, suggesting that the remodeling of PUFAs enhanced inflammatory response. These studies supported the rapid operation of the Lands cycle to maintain cell growth and viability by populating PUFA species; however, without sufficient n-6 fatty acids, 20:3 n-9 accumulated, resulting in altered lipid mediator biosynthesis and inflammatory response. Copyright © 2018 by the American Society for Biochemistry and Molecular Biology, Inc.

  1. Establishment of 9L/F344 rat intracerebral glioma model of brain tumor stem cells

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    Zong-yu XIAO

    2015-04-01

    Full Text Available Objective To establish the 9L/F344 rat intracerebral glioma model of brain tumor stem cells.  Methods Rat 9L gliosarcoma stem-like cells were cultured in serum-free suspension. The expression of CD133 and nestin were tested by immunohistochemistry. A total of 48 inbredline male F344 rats were randomly divided into 2 groups, and 9L tumor sphere cells and 9L monolayer cells were respectively implanted into the right caudate nucleus of F344 rats in 2 groups. Survival time was observed and determined using the method of Kaplan-Meier survival analysis. Fourteen days after implantation or when the rats were dying, their brains were perfused and sectioned for HE staining, and CD133 and nestin were detected by immunohistochemistry.  Results Rat 9L tumor spheres were formed with suspension culture in serum-free medium. The gliomas formed in both groups were invasive without obvious capsule. More new vessels, bleeding and necrosis could be detected in 9L tumor spheres group. The tumor cells in both groups were positive for CD133 and nestin. There was no significant difference in the expression of CD133 and nestin between 2 groups (P > 0.05, for all. According to the expression of nestin, the tumors formed by 9L tumor sphere cells were more invasive. The median survival time of the rats bearing 9L tumor sphere cells was 15 d (95%CI: 15.219-15.781, and the median survival time of the rats bearing 9L monolayer cells was 21 d (95%CI: 20.395-21.605. There was significant difference between 2 groups (χ2 = 12.800, P = 0.000.  Conclusions 9L/F344 rat intracerebral glioma model of brain tumor stem cells is successfully established, which provides a glioma model for the future research. DOI: 10.3969/j.issn.1672-6731.2015.04.012

  2. The dance of the perivascular and endothelial cells: mechanisms of brain response to immune signaling.

    Science.gov (United States)

    Saper, Clifford B

    2010-01-14

    The mechanisms underlying the brain response to systemic inflammation remain unclear. In this issue of Neuron, Serrats and colleagues demonstrate that two cell types that produce prostaglandins that act on the brain, perivascular and endothelial cells, have an unexpectedly complex interaction in regulating the timing and types of brain responses that occur.

  3. Mammary Gland Cell Culture of Macaca fascicularis as a Reservoir for Stem Cells

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    Silmi Mariya

    2017-07-01

    Full Text Available The mammary gland contains adult stem cells that are capable of self-renewal and are likely target for neoplastic transformation leading to breast cancer. In this study, we developed a cell culture derived from the mammary glands of cynomolgus monkeys (Macaca fascicularis (MfMC and furthermore identified the expression of markers for stemness and estrogen receptor-associated activities. We found that the primary culture can be successfully subcultured to at least 3 passages, primarily epithelial-like in morphology, the cultured cells remained heterogenous in phenotype as they expressed epithelial cell markers CD24, CK18, and marker for fibroblast S1004A. Importantly, the cell population also consistently expressed the markers of mammary stem cells (ITGB1 or CD29 and ITGA6 or CD49f, mesenchymal stem cells (CD73 and CD105 and pluripotency (NANOG, OCT4, SOX2. In addition to this, the cells were also positive for Estrogen Receptor (ER, and ER-activated marker Trefoil Factor 1, suggesting an estrogen responsiveness of the culture model. These results indicate that our cell culture model is a reliable model for acquiring a population of cells with mammary stem cell properties and that these cultures may also serve as a reservoir from which more purified populations of stem cell populations can be isolated in the future.

  4. Diffusion chamber culture of human peripheral mononuclear cells in mice

    International Nuclear Information System (INIS)

    Kawakami, Masahito; Shigeta, Chiharu; Enzan, Hideaki; Takahashi, Hiroshi; Ohkita, Takeshi

    1977-01-01

    The mononuclear cells isolated by Isopaque-Ficoll method from blood of three healthy men were cultured in diffusion chambers implanted to peritoneal cavity of mice pretreated by cyclophosphamide 300 mg/kg b. w. or 800 rad of 60 Co γ ray or 500 rad. For two of three men the cell growth was slightly higher in hosts pretreated by cyclophosphamide or 800 rad than in hosts pretreated by 500 rad, but, for another one it was slow in all hosts. Under these conditions the growth potential of mononuclear cells might be different from person to person. A few granulocytes as well as plasma cells, very few megakaryocytes and rare erythroblasts were found in diffusion chamber cultured cells. (auth.)

  5. Microfluidic cell culture chip with multiplexed medium delivery and efficient cell/scaffold loading mechanisms for high-throughput perfusion 3-dimensional cell culture-based assays.

    Science.gov (United States)

    Huang, Song-Bin; Wu, Min-Hsien; Wang, Shih-Siou; Lee, Gwo-Bin

    2011-06-01

    This study reports a microfluidic cell culture chip consisting of 48 microbioreactors for high-throughput perfusion 3-dimensional (3-D) cell culture-based assays. Its advantages include the capability for multiplexed and backflow-free medium delivery, and both efficient and high-throughput micro-scale, 3-D cell culture construct loading. In this work, the microfluidic cell culture chip is fabricated using two major processes, specifically, a computer-numerical-controlled (CNC) mold machining process and a polydimethylsiloxane (PDMS) replication process. The chip is composed of micropumps, microbioreactors, connecting microchannels and a cell/agarose scaffold loading mechanism. The performance of the new pneumatic micropumps and the cell/agarose scaffold loading mechanism has been experimentally evaluated. The experimental results show that this proposed multiplexed medium-pumping design is able to provide a uniform pumping rate ranging from 1.5 to 298.3 μl hr(-1) without any fluid backflow and the resultant medium contamination. In addition, the simple cell/agarose loading method has been proven to be able to load the 3-D cell culture construct uniformly and efficiently in all 48 microbioreactors investigated. Furthermore, a micro-scale, perfusion, 3-D cell culture-based assay has been successfully demonstrated using this proposed cell culture chip. The experimental results are also compared to a similar evaluation using a conventional static 3-D cell culture with a larger scale culture. It is concluded that the choice of a cell culture format can influence assay results. As a whole, because of the inherent advantages of a miniaturized perfusion 3-D cell culture assay, the cell culture chip not only can provide a stable, well-defined and more biologically-meaningful culture environment, but it also features a low consumption of research resources. Moreover, due to the integrated medium pumping mechanism and the simple cell/agarose loading method, this chip is

  6. Associative memory cells and their working principle in the brain

    Science.gov (United States)

    Wang, Jin-Hui; Cui, Shan

    2018-01-01

    The acquisition, integration and storage of exogenous associated signals are termed as associative learning and memory. The consequences and processes of associative thinking and logical reasoning based on these stored exogenous signals can be memorized as endogenous signals, which are essential for decision making, intention, and planning. Associative memory cells recruited in these primary and secondary associative memories are presumably the foundation for the brain to fulfill cognition events and emotional reactions in life, though the plasticity of synaptic connectivity and neuronal activity has been believed to be involved in learning and memory. Current reports indicate that associative memory cells are recruited by their mutual synapse innervations among co-activated brain regions to fulfill the integration, storage and retrieval of associated signals. The activation of these associative memory cells initiates information recall in the mind, and the successful activation of their downstream neurons endorses memory presentations through behaviors and emotion reactions. In this review, we aim to draw a comprehensive diagram for associative memory cells, working principle and modulation, as well as propose their roles in cognition, emotion and behaviors. PMID:29487741

  7. Lingual Epithelial Stem Cells and Organoid Culture of Them

    Directory of Open Access Journals (Sweden)

    Hiroko Hisha

    2016-01-01

    Full Text Available As tongue cancer is one of the major malignant cancers in the world, understanding the mechanism of maintenance of lingual epithelial tissue, which is known to be the origin of tongue cancer, is unquestionably important. However, the actual stem cells that are responsible for the long-term maintenance of the lingual epithelium have not been identified. Moreover, a simple and convenient culture method for lingual epithelial stem cells has not yet been established. Recently, we have shown that Bmi1-positive cells, residing at the second or third layer of the epithelial cell layer at the base of the interpapillary pit (IPP, were slow-cycling and could supply keratinized epithelial cells for over one year, indicating that Bmi1-positive cells are long-term lingual epithelial stem cells. In addition, we have developed a novel lingual epithelium organoid culture system using a three-dimensional matrix and growth factors. Here, we discuss current progress in the identification of lingual stem cells and future applications of the lingual culture system for studying the regulatory mechanisms of the lingual epithelium and for regenerative medicine.

  8. Crude subcellular fractionation of cultured mammalian cell lines

    Directory of Open Access Journals (Sweden)

    Holden Paul

    2009-12-01

    Full Text Available Abstract Background The expression and study of recombinant proteins in mammalian culture systems can be complicated during the cell lysis procedure by contaminating proteins from cellular compartments distinct from those within which the protein of interest resides and also by solubility issues that may arise from the use of a single lysis buffer. Partial subcellular fractionation using buffers of increasing stringency, rather than whole cell lysis is one way in which to avoid or reduce this contamination and ensure complete recovery of the target protein. Currently published protocols involve time consuming centrifugation steps which may require expensive equipment and commercially available kits can be prohibitively expensive when handling large or multiple samples. Findings We have established a protocol to sequentially extract proteins from cultured mammalian cells in fractions enriched for cytosolic, membrane bound organellar, nuclear and insoluble proteins. All of the buffers used can be made inexpensively and easily and the protocol requires no costly equipment. While the method was optimized for a specific cell type, we demonstrate that the protocol can be applied to a variety of commonly used cell lines and anticipate that it can be applied to any cell line via simple optimization of the primary extraction step. Conclusion We describe a protocol for the crude subcellular fractionation of cultured mammalian cells that is both straightforward and cost effective and may facilitate the more accurate study of recombinant proteins and the generation of purer preparations of said proteins from cell extracts.

  9. Treatment of mycoplasma contamination in cell cultures with Plasmocin.

    Science.gov (United States)

    Uphoff, Cord C; Denkmann, Sabine-A; Drexler, Hans G

    2012-01-01

    A high percentage of cell lines are chronically infected with various mycoplasma species. The addition of antibiotics that are particularly effective against these contaminants to the culture medium during a limited period of time is a simple, inexpensive, and very practical approach for decontaminating cell cultures. Here, we examined the effectiveness of the new antimycoplasma compound Plasmocin that has been employed routinely to cleanse chronically infected cell lines. In a first round of treatment 45 out of 58 (78%) mycoplasma-positive cell lines could be cured. In a second attempt using back-up cryopreserved original cells, four additional cell lines were cured; thus, the overall cure rate was 84%. Even if the mycoplasma contamination was not eradicated by Plasmocin, the parallel treatment with several other antibiotics (Baytril, BM-Cyclin, Ciprobay, MRA, or MycoZap) led to the cure of all 58 cell lines. The successful decontamination was permanent as mycoplasmas were no longer detected at day +14 posttreatment and at later time points as examined by PCR which is the most sensitive and specific mycoplasma detection method. Collectively, our results highlight certain antibiotics as effective antimycoplasma reagents and support the therapeutic rationale for their use in the eradication of this notorious cell culture contaminant.

  10. Treatment of Mycoplasma Contamination in Cell Cultures with Plasmocin

    Directory of Open Access Journals (Sweden)

    Cord C. Uphoff

    2012-01-01

    Full Text Available A high percentage of cell lines are chronically infected with various mycoplasma species. The addition of antibiotics that are particularly effective against these contaminants to the culture medium during a limited period of time is a simple, inexpensive, and very practical approach for decontaminating cell cultures. Here, we examined the effectiveness of the new antimycoplasma compound Plasmocin that has been employed routinely to cleanse chronically infected cell lines. In a first round of treatment 45 out of 58 (78% mycoplasma-positive cell lines could be cured. In a second attempt using back-up cryopreserved original cells, four additional cell lines were cured; thus, the overall cure rate was 84%. Even if the mycoplasma contamination was not eradicated by Plasmocin, the parallel treatment with several other antibiotics (Baytril, BM-Cyclin, Ciprobay, MRA, or MycoZap led to the cure of all 58 cell lines. The successful decontamination was permanent as mycoplasmas were no longer detected at day +14 posttreatment and at later time points as examined by PCR which is the most sensitive and specific mycoplasma detection method. Collectively, our results highlight certain antibiotics as effective antimycoplasma reagents and support the therapeutic rationale for their use in the eradication of this notorious cell culture contaminant.

  11. Bridging the gap between cell culture and live tissue

    Directory of Open Access Journals (Sweden)

    Stefan Przyborski

    2017-11-01

    Full Text Available Traditional in vitro two-dimensional (2-D culture systems only partly imitate the physiological and biochemical features of cells in their original tissue. In vivo, in organs and tissues, cells are surrounded by a three-dimensional (3-D organization of supporting matrix and neighbouring cells, and a gradient of chemical and mechanical signals. Furthermore, the presence of blood flow and mechanical movement provides a dynamic environment (Jong et al., 2011. In contrast, traditional in vitro culture, carried out on 2-D plastic or glass substrates, typically provides a static environment, which, however is the base of the present understanding of many biological processes, tissue homeostasis as well as disease. It is clear that this is not an exact representation of what is happening in vivo and the microenvironment provided by in vitro cell culture models are significantly different and can cause deviations in cell response and behaviour from those distinctive of in vivo tissues. In order to translate the present basic knowledge in cell control, cell repair and regeneration from the laboratory bench to the clinical application, we need a better understanding of the cell and tissue interactions. This implies a detailed comprehension of the natural tissue environment, with its organization and local signals, in order to more closely mimic what happens in vivo, developing more physiological models for efficient in vitro systems. In particular, it is imperative to understand the role of the environmental cues which can be mainly divided into those of a chemical and mechanical nature.

  12. [In vitro cell culture technology in cosmetology research].

    Science.gov (United States)

    Gojniczek, Katarzyna; Garncarczyk, Agnieszka; Pytel, Agata

    2005-01-01

    For ages the humanity has been looking for all kind of active substances, which could be used in improving the health and the appearance of our skin. People try to find out how to protect the skin from harmful, environmental factors. Every year a lot of new natural and synthetic, chemical substances are discovered. All of them potentially could be used as a cosmetic ingredient. In cosmetology research most of new xenobiotics were tested in vivo on animals. Alternative methods to in vivo tests are in vitro tests with skin cell culture system. The aim of this work was to describe two-dimensional and tree-dimensional skin cell cultures. Additionally, in this work we wanted to prove the usefulness of in vitro skin cell cultures in cosmetology research.

  13. Children's brain tumour cells produce RNA particles with incomplete retrovirus characteristics.

    Science.gov (United States)

    Thorne, H J; Jose, D G; Adams, F C; Coelen, R J; Whitehead, R H; Klugg, G

    1982-01-01

    Short-term cultures of cells from human rain tumours have been reported to synthesise RNA particles of density in the range characteristic of C type RNA retroviruses, with associated DNA polymerase activity. Fresh tumour cells obtained from 6 children with astrocytoma and 7 children with medulloblastoma, together with one sample of normal brain tissue and normal leukocytes from brain tumour patients were assayed by several characteristics for the primate retrovirus. 1 or 6 (17%) astrocytomas and 4 of 7 (57%) medulloblastomas released RNA particles which banded in sucrose gradients at a density of 1.16-1.18 g/cm3 together with a short segment of DNA, which was eliminated by prior ribonuclease treatment and two proteins of 28k and 16k daltons. These findings were compatible with the presence of a primate retrovirus. Immune coprecipitation of 125I-labelled proteins from the 1.16-1.18 g/cm3 gradient region failed to show any reactivity with antisera to p28 core antigens or the p70 reverse transcriptase antigens of simian sarcoma virus, baboon endogenous virus or Mason Pfizer virus. Assays for DNA polymerase activity in culture supernatant fluid showed only a low amount of activity with template preferences not characteristic of the retroviral reverse transcriptase enzyme.

  14. Isolation, culture and genetic manipulation of mouse pancreatic ductal cells.

    Science.gov (United States)

    Reichert, Maximilian; Takano, Shigetsugu; Heeg, Steffen; Bakir, Basil; Botta, Gregory P; Rustgi, Anil K

    2013-01-01

    The most common subtype of pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC). PDAC resembles duct cells morphologically and, to some extent, at a molecular level. Recently, genetic-lineage labeling has become popular in the field of tumor biology in order to study cell-fate decisions or to trace cancer cells in the mouse. However, certain biological questions require a nongenetic labeling approach to purify a distinct cell population in the pancreas. Here we describe a protocol for isolating mouse pancreatic ductal epithelial cells and ductlike cells directly in vivo using ductal-specific Dolichos biflorus agglutinin (DBA) lectin labeling followed by magnetic bead separation. Isolated cells can be cultured (in two or three dimensions), manipulated by lentiviral transduction to modulate gene expression and directly used for molecular studies. This approach is fast (~4 h), affordable, results in cells with high viability, can be performed on the bench and is applicable to virtually all genetic and nongenetic disease models of the pancreas.

  15. DNA synthesis and cell division in the adult primate brain

    International Nuclear Information System (INIS)

    Rakic, P.

    1985-01-01

    It is generally accepted that the adult human brain is incapable of producing new neuron. Even cursory examination of neurologic, neuropathologic, or neurobiological textbooks published during the past 50 years will testify that this belief is deeply entrenched. In his classification of cell populations on the basis of their proliferative behavior, Leblond regarded neurons of the central nervous system as belonging to a category of static, nonrenewing epithelial tissue incapable of expanding or replenishing itself. This belief, however needs to re reexamined for two major reasons: First, as reviewed below, a number of reports have provided evidence of neurogenesis in adult brain of several vertebrate species. Second, the capacity for neurogenesis in the adult primate central nervous system has never been examined by modern methods. In this article the author described recent results from an extensive autoradiographic analysis performed on twelve rhesus monkeys injected with the specific DNA precursor [ 3 H] thymidine at ages ranging from 6 postnatal months to 17 years

  16. Aragonite precipitation by "proto-polyps" in coral cell cultures.

    Science.gov (United States)

    Mass, Tali; Drake, Jeana L; Haramaty, Liti; Rosenthal, Yair; Schofield, Oscar M E; Sherrell, Robert M; Falkowski, Paul G

    2012-01-01

    The mechanisms of coral calcification at the molecular, cellular and tissue levels are poorly understood. In this study, we examine calcium carbonate precipitation using novel coral tissue cultures that aggregate to form "proto-polyps". Our goal is to establish an experimental system in which calcification is facilitated at the cellular level, while simultaneously allowing in vitro manipulations of the calcifying fluid. This novel coral culturing technique enables us to study the mechanisms of biomineralization and their implications for geochemical proxies. Viable cell cultures of the hermatypic, zooxanthellate coral, Stylophora pistillata, have been maintained for 6 to 8 weeks. Using an enriched seawater medium with aragonite saturation state similar to open ocean surface waters (Ω(arag)~4), the primary cell cultures assemble into "proto-polyps" which form an extracellular organic matrix (ECM) and precipitate aragonite crystals. These extracellular aragonite crystals, about 10 µm in length, are formed on the external face of the proto-polyps and are identified by their distinctive elongated crystallography and X-ray diffraction pattern. The precipitation of aragonite is independent of photosynthesis by the zooxanthellae, and does not occur in control experiments lacking coral cells or when the coral cells are poisoned with sodium azide. Our results demonstrate that proto-polyps, aggregated from primary coral tissue culture, function (from a biomineralization perspective) similarly to whole corals. This approach provides a novel tool for investigating the biophysical mechanism of calcification in these organisms.

  17. Aragonite precipitation by "proto-polyps" in coral cell cultures.

    Directory of Open Access Journals (Sweden)

    Tali Mass

    Full Text Available The mechanisms of coral calcification at the molecular, cellular and tissue levels are poorly understood. In this study, we examine calcium carbonate precipitation using novel coral tissue cultures that aggregate to form "proto-polyps". Our goal is to establish an experimental system in which calcification is facilitated at the cellular level, while simultaneously allowing in vitro manipulations of the calcifying fluid. This novel coral culturing technique enables us to study the mechanisms of biomineralization and their implications for geochemical proxies. Viable cell cultures of the hermatypic, zooxanthellate coral, Stylophora pistillata, have been maintained for 6 to 8 weeks. Using an enriched seawater medium with aragonite saturation state similar to open ocean surface waters (Ω(arag~4, the primary cell cultures assemble into "proto-polyps" which form an extracellular organic matrix (ECM and precipitate aragonite crystals. These extracellular aragonite crystals, about 10 µm in length, are formed on the external face of the proto-polyps and are identified by their distinctive elongated crystallography and X-ray diffraction pattern. The precipitation of aragonite is independent of photosynthesis by the zooxanthellae, and does not occur in control experiments lacking coral cells or when the coral cells are poisoned with sodium azide. Our results demonstrate that proto-polyps, aggregated from primary coral tissue culture, function (from a biomineralization perspective similarly to whole corals. This approach provides a novel tool for investigating the biophysical mechanism of calcification in these organisms.

  18. Parents' experiences following children's moderate to severe traumatic brain injury: a clash of cultures.

    Science.gov (United States)

    Roscigno, Cecelia I; Swanson, Kristen M

    2011-10-01

    Little is understood about parents' experiences following children's moderate to severe traumatic brain injury (TBI). Using descriptive phenomenology, we explored common experiences of parents whose children were diagnosed with moderate to severe TBI. Parents from across the United States (N = 42, from 37 families) participated in two semistructured interviews (~ 90 minutes in length and 12 to 15 months apart) in the first 5 years following children's TBI. First interviews were in person. Second interviews, done in person or by phone, facilitated updating parents' experiences and garnering their critique of the descriptive model. Parent themes were (a) grateful to still have my child, (b) grieving for the child I knew, (c) running on nerves, and (d) grappling to get what my child and family need. Parents reported cultural barriers because of others' misunderstandings. More qualitative inquiry is needed to understand how the knowledge, attitudes, beliefs, and culture-based expectations of others influence parents' interactions and the family's adjustment and well-being.

  19. Karyotype changes in cultured human corneal endothelial cells

    OpenAIRE

    Miyai, Takashi; Maruyama, Yoko; Osakabe, Yasuhiro; Nejima, Ryohei; Miyata, Kazunori; Amano, Shiro

    2008-01-01

    Purpose To examine karyotype changes in cultured human corneal endothelial cells (HCECs). Methods HCECs with Descemet’s membrane were removed from 20 donors of various ages (range, 2–77 years; average, 43.7±26.4 years) and cultured on dishes coated with extracellular matrix produced by bovine corneal endothelial cells (BCECs). Karyotype changes were examined by G-band karyotyping of HCECs at the third passage from 12 donors and the fifth passage from 16 donors. The number of chromosomes was a...

  20. A deficit in zinc availability can cause alterations in tubulin thiol redox status in cultured neurons and in the developing fetal rat brain.

    Science.gov (United States)

    Mackenzie, Gerardo G; Salvador, Gabriela A; Romero, Carolina; Keen, Carl L; Oteiza, Patricia I

    2011-07-15

    Zinc (Zn) deficiency during early development can result in multiple brain abnormalities and altered neuronal functions. In rats, a gestational deficit of Zn can affect the fetal brain cytoskeleton and signaling cascades involved in cellular processes that are central to brain development. In this paper, we tested the hypothesis that oxidative stress is involved in Zn deficiency-induced altered tubulin dynamics and the associated dysregulation of transcription factor NF-κB. For this purpose, we used two cell culture models (rat cortical neurons, human IMR-32 neuroblastoma cells) and an animal model of Zn deficiency. A low rate of in vitro tubulin polymerization, an increase in tubulin oligomers, and a higher protein cysteine oxidation were observed in the Zn-deficient neuronal cells and in gestation day 19 fetal brains obtained from dams fed marginal-Zn diets throughout pregnancy. These alterations could be prevented by treating the Zn-deficient cells with the reducing agent tris(2-carboxyethyl)phosphine or by the presence of N-acetylcysteine (NAC) and α-lipoic acid (LA). Consistent with the above, Zn deficiency-induced tubulin-mediated alterations in transcription factor NF-κB nuclear translocation were prevented by treating IMR-32 cells with LA and NAC. Binding of the NF-κB protein p50, dynein, and karyopherin α (components of the NF-κB transport complex) to β-tubulin as well as the expression of NF-κB-dependent genes (Bcl-2, cyclin D1, and c-myc) was also restored by the addition of LA and NAC to Zn-deficient cells. In conclusion, a deficit in Zn viability could affect early brain development through: (1) an induction of oxidative stress, (2) tubulin oxidation, (3) altered tubulin dynamics, and (4) deregulation of signals (e.g., NF-κB) involved in critical developmental events. Copyright © 2011 Elsevier Inc. All rights reserved.

  1. Testicular Sertoli cells influence the proliferation and immunogenicity of co-cultured endothelial cells

    International Nuclear Information System (INIS)

    Fan, Ping; He, Lan; Pu, Dan; Lv, Xiaohong; Zhou, Wenxu; Sun, Yining; Hu, Nan

    2011-01-01

    Research highlights: → The proliferation of dramatic increased by co-cultured with Sertoli cells. → VEGF receptor-2 expression of ECs was up-regulated by co-cultured with Sertoli cells. → The MHC expression of ECs induced by INF-γ and IL-6, IL-8 and sICAM induced by TNF-α decreased respectively after co-cultured with Sertoli cells. → ECs co-cultured with Sertoli cells also didn't increase the stimulation index of spleen lymphocytes. -- Abstract: The major problem of the application of endothelial cells (ECs) in transplantation is the lack of proliferation and their immunogenicity. In this study, we co-cultured ECs with Sertoli cells to monitor whether Sertoli cells can influence the proliferation and immunogenicity of co-cultured ECs. Sertoli cells were isolated from adult testicular tissue. ECs were divided into the control group and the experimental group, which included three sub-groups co-cultured with 1 x 10 3 , 1 x 10 4 or 1 x 10 5 cell/ml of Sertoli cells. The growth and proliferation of ECs were observed microscopically, and the expression of vascular endothelial growth factor (VEGF) receptor-2 (KDR) was examined by Western blotting. In another experiment, ECs were divided into the control group, the single culture group and the co-culture group with the optimal concentration of Sertoli cells. After INF-γ and TNF-α were added to the culture medium, MHC II antigen expression was detected by immunofluorescence staining and western blotting; interleukin (IL)-6, IL-8 and soluble intercellular adhesion molecule (sICAM) were measured in the culture medium by ELISA. We demonstrated that 1 x 10 4 cell/ml Sertoli cells promoted the proliferation of co-cultured ECs more dramatically than that in other groups (P 4 cell/ml of the Sertoli cells was most effective in the up-regulation of KDR expression in the co-cultured ECs (P < 0.05). Sertoli cells can effectively suppress INF-γ-induced MHC II antigen expression in co-cultured ECs compared with single

  2. Testicular Sertoli cells influence the proliferation and immunogenicity of co-cultured endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Fan, Ping, E-mail: fanpinggoodluck@163.com [Department of Rheumatism and Immunity, The First Affiliated Hospital Xi' an Jiaotong University School of Medicine, Xi' an, Shaanxi 710061 (China); He, Lan; Pu, Dan; Lv, Xiaohong; Zhou, Wenxu; Sun, Yining; Hu, Nan [Department of Rheumatism and Immunity, The First Affiliated Hospital Xi' an Jiaotong University School of Medicine, Xi' an, Shaanxi 710061 (China)

    2011-01-21

    Research highlights: {yields} The proliferation of dramatic increased by co-cultured with Sertoli cells. {yields} VEGF receptor-2 expression of ECs was up-regulated by co-cultured with Sertoli cells. {yields} The MHC expression of ECs induced by INF-{gamma} and IL-6, IL-8 and sICAM induced by TNF-{alpha} decreased respectively after co-cultured with Sertoli cells. {yields} ECs co-cultured with Sertoli cells also didn't increase the stimulation index of spleen lymphocytes. -- Abstract: The major problem of the application of endothelial cells (ECs) in transplantation is the lack of proliferation and their immunogenicity. In this study, we co-cultured ECs with Sertoli cells to monitor whether Sertoli cells can influence the proliferation and immunogenicity of co-cultured ECs. Sertoli cells were isolated from adult testicular tissue. ECs were divided into the control group and the experimental group, which included three sub-groups co-cultured with 1 x 10{sup 3}, 1 x 10{sup 4} or 1 x 10{sup 5} cell/ml of Sertoli cells. The growth and proliferation of ECs were observed microscopically, and the expression of vascular endothelial growth factor (VEGF) receptor-2 (KDR) was examined by Western blotting. In another experiment, ECs were divided into the control group, the single culture group and the co-culture group with the optimal concentration of Sertoli cells. After INF-{gamma} and TNF-{alpha} were added to the culture medium, MHC II antigen expression was detected by immunofluorescence staining and western blotting; interleukin (IL)-6, IL-8 and soluble intercellular adhesion molecule (sICAM) were measured in the culture medium by ELISA. We demonstrated that 1 x 10{sup 4} cell/ml Sertoli cells promoted the proliferation of co-cultured ECs more dramatically than that in other groups (P < 0.05). Western blotting showed that 1 x 10{sup 4} cell/ml of the Sertoli cells was most effective in the up-regulation of KDR expression in the co-cultured ECs (P < 0.05). Sertoli

  3. A study of chromosomal aberrations in amniotic fluid cell cultures.

    Science.gov (United States)

    Wolstenholme, J; Crocker, M; Jonasson, J

    1988-06-01

    This paper represents the analysis of 1916 routine amniotic fluid specimens harvested by an in situ fixation technique in a prospective study with regard to cultural chromosome anomalies. Excluding constitutional abnormalities, 2.9 per cent of 19,432 cells analysed showed some form of chromosome anomaly, terminal deletions (57 per cent) and chromatid/chromosome breaks and gaps (18 per cent) being the most frequent, followed by interchange aberrations (13 per cent) and trisomy (5 per cent). No case was found of more than one colony from the same culture showing the same anomaly without it being present in other cultures from the same fluid. The wholly abnormal colonies had a surplus of trisomies and from the mathematical considerations presented one may infer that these are likely to reflect the presence of abnormal cells in the amniotic fluid. Partly abnormal colonies appeared at a frequency that would correspond to virtual absence of selection against chromosomally abnormal cells when cultured in vitro. The aberrations found were similar to those seen as single cell anomalies, except for chromatid breaks and exchanges. The data suggest a basic preferential induction of trisomy for chromosomes 2, 18, 21, and the Y-chromosome. Structural aberrations showed a marked clustering of breakpoints around the centromeres. The frequency of mutant cells was low (1.4 X 10(-3)) before culture was initiated. At harvest, the frequency of abnormal cells was much higher (3 X 10(-2)) corresponding to 3 X 10(-3) mutations per cell per generation accumulating over approximately ten generations in vitro.

  4. Cell culture media impact on drug product solution stability.

    Science.gov (United States)

    Purdie, Jennifer L; Kowle, Ronald L; Langland, Amie L; Patel, Chetan N; Ouyang, Anli; Olson, Donald J

    2016-07-08

    To enable subcutaneous administration of monoclonal antibodies, drug product solutions are often needed at high concentrations. A significant risk associated with high drug product concentrations is an increase in aggregate level over the shelf-life dating period. While much work has been done to understand the impact of drug product formulation on aggregation, there is limited understanding of the link between cell culture process conditions and soluble aggregate growth in drug product. During cell culture process development, soluble aggregates are often measured at harvest using cell-free material purified by Protein A chromatography. In the work reported here, cell culture media components were evaluated with respect to their impact on aggregate levels in high concentration solution drug product during accelerated stability studies. Two components, cysteine and ferric ammonium citrate, were found to impact aggregate growth rates in our current media (version 1) leading to the development of new chemically defined media and concentrated feed formulations. The new version of media and associated concentrated feeds (version 2) were evaluated across four cell lines producing recombinant IgG4 monoclonal antibodies and a bispecific antibody. In all four cell lines, the version 2 media reduced aggregate growth over the course of a 12 week accelerated stability study compared with the version 1 media, although the degree to which aggregate growth decreased was cell line dependent. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:998-1008, 2016. © 2016 American Institute of Chemical Engineers.

  5. Testing of serum atherogenicity in cell cultures: questionable data published

    Directory of Open Access Journals (Sweden)

    Sergei V. Jargin

    2012-01-01

    Full Text Available In a large series of studies was reported that culturing of smooth muscle cells with serum from atherosclerosis patients caused intracellular lipid accumulation, while serum from healthy controls had no such effect. Cultures were used for evaluation of antiatherogenic drugs. Numerous substances were reported to lower serum atherogenicity: statins, trapidil, calcium antagonists, garlic derivatives etc. On the contrary, beta-blockers, phenothiazines and oral hypoglycemics were reported to be pro-atherogenic. Known antiatherogenic agents can influence lipid metabolism and cholesterol synthesis, intestinal absorption or endothelium-related mechanisms. All these targets are absent in cell monocultures. Inflammatory factors, addressed by some antiatherogenic drugs, are also not reproduced. In vivo, relationship between cholesterol uptake by cells and atherogenesis must be inverse rather than direct: in familial hypercholesterolemia, inefficient clearance of LDL-cholesterol by cells predisposes to atherosclerosis. Accordingly, if a pharmacological agent reduces cholesterol uptake by cells in vitro, it should be expected to elevate cholesterol in vivo. Validity of clinical recommendations, based on serum atherogenicity testing in cell monocultures, is therefore questionable. These considerations pertain also to the drugs developed on the basis of the cell culture experiments.

  6. Arsenic exposure induces the Warburg effect in cultured human cells

    International Nuclear Information System (INIS)

    Zhao, Fei; Severson, Paul; Pacheco, Samantha; Futscher, Bernard W.; Klimecki, Walter T.

    2013-01-01

    Understanding how arsenic exacts its diverse, global disease burden is hampered by a limited understanding of the particular biological pathways that are disrupted by arsenic and underlie pathogenesis. A reductionist view would predict that a small number of basic pathways are generally perturbed by arsenic, and manifest as diverse diseases. Following an initial observation that arsenite-exposed cells in culture acidify their media more rapidly than control cells, the report here shows that low level exposure to arsenite (75 ppb) is sufficient to induce aerobic glycolysis (the Warburg effect) as a generalized phenomenon in cultured human primary cells and cell lines. Expanded studies in one such cell line, the non-malignant pulmonary epithelial line, BEAS-2B, established that the arsenite-induced Warburg effect was associated with increased accumulation of intracellular and extracellular lactate, an increased rate of extracellular acidification, and inhibition by the non-metabolized glucose analog, 2-deoxy-D-glucose. Associated with the induction of aerobic glycolysis was a pathway-wide induction of glycolysis gene expression, as well as protein accumulation of an established glycolysis master-regulator, hypoxia-inducible factor 1A. Arsenite-induced alteration of energy production in human cells represents the type of fundamental perturbation that could extend to many tissue targets and diseases. - Highlights: • Chronic arsenite exposure induces aerobic glycolysis, dubbed the “Warburg effect”. • Arsenite-induced Warburg effect is a general phenomenon in cultured human cells. • HIF-1A may mediate arsenite induced Warburg effect

  7. Scanning electroporation of selected areas of adherent cell cultures.

    Science.gov (United States)

    Olofsson, Jessica; Levin, Mikael; Strömberg, Anette; Weber, Stephen G; Ryttsén, Frida; Orwar, Owe

    2007-06-15

    We present a computer-controlled scanning electroporation method. Adherent cells are electroporated using an electrolyte-filled capillary in contact with an electrode. The capillary can be scanned over a cell culture and locally deliver both an electric field and an electroporation agent to the target area without affecting surrounding cells. The instantaneous size of the targeted area is determined by the dimensions of the capillary. The size and shape of the total electroporated area are defined by these dimensions in combination with the scanning pattern. For example, striped and serpentine patterns of electroporated cells in confluent cultures can be formed. As it is easy to switch between different electroporation agents, the method is suitable for design of cell cultures with complex composition. Finite element method simulations were used to study the spatial distributions of the electric field and the concentration of an electroporation agent, as well as the fluid dynamics related to scanning and flow of electroporation agent from the capillary. The method was validated for transfection by introduction of a 9-base-pair-long randomized oligonucleotide into PC12 cells and a pmaxGFP plasmid coding for green fluorescent protein into CHO and WSS cells.

  8. Schwann Cells Can Be Reprogrammed to Multipotency by Culture

    Science.gov (United States)

    Widera, Darius; Heimann, Peter; Zander, Christin; Imielski, Yvonne; Heidbreder, Meike; Heilemann, Mike; Kaltschmidt, Christian

    2011-01-01

    Adult neural crest related-stem cells persist in adulthood, making them an ideal and easily accessible source of multipotent cells for potential clinical use. Recently, we reported the presence of neural crest-related stem cells within adult palatal ridges, thus raising the question of their localization in their endogenous niche. Using immunocytochemistry, reverse transcription–polymerase chain reaction, and correlative fluorescence and transmission electron microscopy, we identified myelinating Schwann cells within palatal ridges as a putative neural crest stem cell source. Palatal Schwann cells expressed nestin, p75NTR, and S100. Correlative fluorescence and transmission electron microscopy revealed the exclusive nestin expression within myelinating Schwann cells. Palatal neural crest stem cells and nestin-positive Schwann cells isolated from adult sciatic nerves were able to grow under serum-free conditions as neurospheres in presence of FGF-2 and EGF. Spheres of palatal and sciatic origin showed overlapping expression pattern of neural crest stem cell and Schwann cell markers. Expression of the pluripotency factors Sox2, Klf4, c-Myc, Oct4, the NF-κB subunits p65, p50, and the NF-κB-inhibitor IκB-β were up-regulated in conventionally cultivated sciatic nerve Schwann cells and in neurosphere cultures. Finally, neurospheres of palatal and sciatic origin were able to differentiate into ectodermal, mesodermal, and endodermal cell types emphasizing their multipotency. Taken together, we show that nestin-positive myelinating Schwann cells can be reprogrammed into multipotent adult neural crest stem cells under appropriate culture conditions. PMID:21466279

  9. Glial cells as progenitors and stem cells: new roles in the healthy and diseased brain.

    Science.gov (United States)

    Dimou, Leda; Götz, Magdalena

    2014-07-01

    The diverse functions of glial cells prompt the question to which extent specific subtypes may be devoted to a specific function. We discuss this by reviewing one of the most recently discovered roles of glial cells, their function as neural stem cells (NSCs) and progenitor cells. First we give an overview of glial stem and progenitor cells during development; these are the radial glial cells that act as NSCs and other glial progenitors, highlighting the distinction between the lineage of cells in vivo and their potential when exposed to a different environment, e.g., in vitro. We then proceed to the adult stage and discuss the glial cells that continue to act as NSCs across vertebrates and others that are more lineage-restricted, such as the adult NG2-glia, the most frequent progenitor type in the adult mammalian brain, that remain within the oligodendrocyte lineage. Upon certain injury conditions, a distinct subset of quiescent astrocytes reactivates proliferation and a larger potential, clearly demonstrating the concept of heterogeneity with distinct subtypes of, e.g., astrocytes or NG2-glia performing rather different roles after brain injury. These new insights not only highlight the importance of glial cells for brain repair but also their great potential in various aspects of regeneration. Copyright © 2014 the American Physiological Society.

  10. Feline Neural Progenitor Cells I: Long-Term Expansion under Defined Culture Conditions

    Directory of Open Access Journals (Sweden)

    Jing Yang

    2012-01-01

    Full Text Available Neural progenitor cells (NPCs of feline origin (cNPCs have demonstrated utility in transplantation experiments, yet are difficult to grow in culture beyond the 1 month time frame. Here we use an enriched, serum-free base medium (Ultraculture and report the successful long-term propagation of these cells. Primary cultures were derived from fetal brain tissue and passaged in DMEM/F12-based or Ultraculture-based proliferation media, both in the presence of EGF + bFGF. Cells in standard DMEM/F12-based medium ceased to proliferate by 1-month, whereas the cells in the Ultraculture-based medium continued to grow for at least 5 months (end of study with no evidence of senescence. The Ultraculture-based cultures expressed lower levels of progenitor and lineage-associated markers under proliferation conditions but retained multipotency as evidenced by the ability to differentiate into neurons and glia following growth factor removal in the presence of FBS. Importantly, later passage cNPCs did not develop chromosomal aberrations.

  11. Cannabinoids induce incomplete maturation of cultured human leukemia cells

    Energy Technology Data Exchange (ETDEWEB)

    Murison, G.; Chubb, C.B.H.; Maeda, S.; Gemmell, M.A.; Huberman, E.

    1987-08-01

    Monocyte maturation markers were induced in cultured human myeloblastic ML-2 leukemia cells after treatment for 1-6 days with 0.03-30 ..mu..M ..delta../sup 9/-tetrahydrocannabinol (THC), the major psychoactive component of marijuana. After a 2-day or longer treatment, 2- to 5-fold increases were found in the percentages of cells exhibiting reactivity with either the murine OKM1 monoclonal antibody of the Leu-M5 monoclonal antibody, staining positively for nonspecific esterase activity, and displaying a promonocyte morphology. The increases in these differentiation markers after treatment with 0.03-1 ..mu..M THC were dose dependent. At this dose range, THC did not cause an inhibition of cell growth. The THC-induced cell maturation was also characterized by specific changes in the patterns of newly synthesized proteins. The THC-induced differentiation did not, however, result in cells with a highly developed mature monocyte phenotype. However, treatment of these incompletely matured cells with either phorbol 12-myristate 13-acetate of 1..cap alpha..,25-dihydroxycholecalciferol, which are inducers of differentiation in myeloid leukemia cells (including ML-2 cells), produced cells with a mature monocyte morphology. The ML-2 cell system described here may be a useful tool for deciphering critical biochemical events that lead to the cannabinoid-induced incomplete cell differentiation of ML-2 cells and other related cell types. Findings obtained from this system may have important implications for studies of cannabinoid effects on normal human bone-marrow progenitor cells.

  12. Hypofractionated Whole-Brain Radiotherapy for Multiple Brain Metastases From Transitional Cell Carcinoma of the Bladder

    International Nuclear Information System (INIS)

    Rades, Dirk; Meyners, Thekla; Veninga, Theo; Stalpers, Lukas J.A.; Schild, Steven E.

    2010-01-01

    Purpose: Brain metastases in bladder cancer patients are extremely rare. Most patients with multiple lesions receive longer-course whole-brain radiotherapy (WBRT) with 10 x 3 Gy/2 weeks or 20 x 2 Gy/4 weeks. Because its radiosensitivity is relatively low, metastases from bladder cancer may be treated better with hypofractionated radiotherapy. This study compared short-course hypofractionated WBRT (5 x 4 Gy/1 week) to longer-course WBRT. Methods and Materials: Data for 33 patients receiving WBRT alone for multiple brain metastases from transitional cell bladder carcinoma were retrospectively analyzed. Short-course WBRT with 5 x 4 Gy (n = 12 patients) was compared to longer-course WBRT with 10 x 3 Gy/20 x 2 Gy (n = 21 patients) for overall survival (OS) and local (intracerebral) control (LC). Five additional potential prognostic factors were investigated: age, gender, Karnofsky performance score (KPS), number of brain metastases, and extracranial metastases. The Bonferroni correction for multiple tests was used to adjust the p values derived from the multivariate analysis. p values of <0.025 were considered significant. Results: At 6 months, OS was 42% after 5 x 4 Gy and 24% after 10 x 3/20 x 2 Gy (p = 0.31). On univariate analysis, improved OS was associated with less than four brain metastases (p = 0.021) and almost associated with a lack of extracranial metastases (p = 0.057). On multivariate analysis, both factors were not significant. At 6 months, LC was 83% after 5 x 4 Gy and 27% after 10 x 3/20 x 2 Gy (p = 0.035). Improved LC was almost associated with a KPS of ≥70 (p = 0.051). On multivariate analysis, WBRT regimen was almost significant (p = 0.036). KPS showed a trend (p = 0.07). Conclusions: Short-course WBRT with 5 x 4 Gy should be seriously considered for most patients with multiple brain metastases from bladder cancer, as it resulted in improved LC.

  13. Brain pericytes among cells constituting the blood-brain barrier are highly sensitive to tumor necrosis factor-α, releasing matrix metalloproteinase-9 and migrating in vitro

    Directory of Open Access Journals (Sweden)

    Miyaji Haruki

    2011-08-01

    Full Text Available Abstract Background Increased matrix metalloproteinase (MMP-9 in the plasma and brain is associated with blood-brain barrier (BBB disruption through proteolytic activity in neuroinflammatory diseases. MMP-9 is present in the brain microvasculature and its vicinity, where brain microvascular endothelial cells (BMECs, pericytes and astrocytes constitute the BBB. Little is known about the cellular source and role of MMP-9 at the BBB. Here, we examined the ability of pericytes to release MMP-9 and migrate in response to inflammatory mediators in comparison with BMECs and astrocytes, using primary cultures isolated from rat brains. Methods The culture supernatants were collected from primary cultures of rat brain endothelial cells, pericytes, or astrocytes. MMP-9 activities and levels in the supernatants were measured by gelatin zymography and western blot, respectively. The involvement of signaling molecules including mitogen-activated protein kinases (MAPKs and phosphoinositide-3-kinase (PI3K/Akt in the mediation of tumor necrosis factor (TNF-α-induced MMP-9 release was examined using specific inhibitors. The functional activity of MMP-9 was evaluated by a cell migration assay. Results Zymographic and western blot analyses demonstrated that TNF-α stimulated pericytes to release MMP-9, and this release was much higher than from BMECs or astrocytes. Other inflammatory mediators [interleukin (IL-1β, interferon-γ, IL-6 and lipopolysaccharide] failed to induce MMP-9 release from pericytes. TNF-α-induced MMP-9 release from pericytes was found to be mediated by MAPKs and PI3K. Scratch wound healing assay showed that in contrast to BMECs and astrocytes the extent of pericyte migration was significantly increased by TNF-α. This pericyte migration was inhibited by anti-MMP-9 antibody. Conclusion These findings suggest that pericytes are most sensitive to TNF-α in terms of MMP-9 release, and are the major source of MMP-9 at the BBB. This pericyte

  14. System-level modeling and simulation of the cell culture microfluidic biochip ProCell

    DEFF Research Database (Denmark)

    Minhass, Wajid Hassan; Pop, Paul; Madsen, Jan

    2010-01-01

    Microfluidic biochips offer a promising alternative to a conventional biochemical laboratory. There are two technologies for the microfluidic biochips: droplet-based and flow-based. In this paper we are interested in flow-based microfluidic biochips, where the liquid flows continuously through pre......-defined micro-channels using valves and pumps. We present an approach to the system-level modeling and simulation of a cell culture microfluidic biochip called ProCell, Programmable Cell Culture Chip. ProCell contains a cell culture chamber, which is envisioned to run 256 simultaneous experiments (viewed...... and a comprehensive fault model that captures permanent faults occurring during chip operation. Using the proposed modeling and simulation framework, we perform an architectural level evaluation of two cell culture chamber implementations. A qualitative success metric is also proposed to evaluate chip performance...

  15. Chikungunya virus isolation using simplified cell culture technique in Mauritius.

    Science.gov (United States)

    Pyndiah, M N; Pursem, V; Meetoo, G; Daby, S; Ramuth, V; Bhinkah, P; Chuttoo, R; Paratian, U

    2012-03-01

    During the chikungunya outbreak of 2005 - 2006, the only laboratory facilities available in Mauritius were virus isolation in cell culture tubes and serology. The laboratory was submerged with large numbers of blood samples. Comparative isolation was made in human embryonic lung (HEL) and VERO cells grown in 96-well plate. Culture on HEL cells was found to be more sensitive and presence of cytopathic effect (CPE) was observed earlier than in VERO cells. Out of the 18 300 blood samples inoculated on HEL, 11 165 were positive. This virus isolation method was of great help for the surveillance and control of the vectors. In cases of an outbreak a cheap, rapid and simple method of isolating chikungunya virus is described.

  16. Enhancement effect of shikonin in cell suspension culture and transfermanant culture by radiation application

    International Nuclear Information System (INIS)

    Kim, Jae Sung; Lee, Young Keun; Chung, Byung Yeoup; Lee, Young Bok; Hwang Hye Yeon

    2004-10-01

    The cell lines 679, 679-29 and 622-46 of L. erythrorhizon could be selected on LS agar medium for the production shikonin in cell suspension culture. The shikonin was increased moderately in suspension culture of cell line 622-46 in LS liquid medium containing BA 2 mg·L -1 and IAA 0.2 mg·L -1 in the dark, and was increased by adding 1 μM Cu 2+ and 100 μM methyl jasmonate The accumulation of shikonin in the liquid medium was increased significantly by 2 Gy irradiation to callus of cell line 622-46 and culture in LS liquid medium containing BA 2 mg·L -1 and IAA 0.2 mg·L -1 in the dark and shikonin in cell debris was higher by 16 Gy irradiation. The activity of p-hydroxybenzoate geranyltransferase was increased by irradiation of 2 Gy and 16 Gy of γ radiation. Seedling hypocotyles of L. erythrorhizon were infected with Agrogacterium rhizogenes strain 15834 harboring a binary vector with an intron bearing the GUS (β-glucuronidase) gene driven by cauliflower mosaic virus (CaMV) 35S promotor as well as the HPT (hygromycin phosphotransferase) gene as the selection marker. Hairy roots isolated were hygromycin resistant and had integrated GUS gene in DNA. The root tip grown on M-9 medium showed normal pigment production pattern in border cells and root hairs

  17. Magnetic resonance imaging and cell-based neurorestorative therapy after brain injury

    Directory of Open Access Journals (Sweden)

    Quan Jiang

    2016-01-01

    Full Text Available Restorative cell-based therapies for experimental brain injury, such as stroke and traumatic brain injury, substantially improve functional outcome. We discuss and review state of the art magnetic resonance imaging methodologies and their applications related to cell-based treatment after brain injury. We focus on the potential of magnetic resonance imaging technique and its associated challenges to obtain useful new information related to cell migration, distribution, and quantitation, as well as vascular and neuronal remodeling in response to cell-based therapy after brain injury. The noninvasive nature of imaging might more readily help with translation of cell-based therapy from the laboratory to the clinic.

  18. [An experimental study on the Chinese lung adenocarcinoma cell clone CPA-Yang1-BR with brain metastasis potency in nude mice and in vivo imaging research].

    Science.gov (United States)

    Lei, Bei; Cao, Jie; Shen, Jie; Zhao, Lanxiang; Liang, Sheng; Meng, Qinggang; Xie, Wenhui; Yang, Shunfang

    2013-08-20

    Lung cancer is the leading cause of cancer-related death in men and women. It is also the most common cause of brain metastases. A brain metastasis model is difficult to be established because of the presence of the blood-brain barrier (BBB) and the lack of optimal methods for detecting brain metastasis in nude mice. Thus, the establishment of a Chinese lung adenocarcinoma cell line and its animal model with brain metastasis potency and in vivo research is of great significance. CPA-Yang1 cells were obtained from a patient with human lung adenocarcinoma by lentiviral vector-mediated transfection of green fluorescence protein. Intracardiac inoculation of the cells was performed in nude mice, and brain metastatic lesions were detected using micro ¹⁸F FDG-PET/CT scanners, small animal in vivo imaging system for fluorescence, radionuclide and X ray fused imaging, magnetic resonance imaging (MRI) with sense body detection, and resection. The samples were divided into two parts for cell culture and histological diagnosis. The process was repeated in vivo and in vitro for four cycles to obtain a novel cell clone, CPA-Yang1-BR. A novel cell clone, CPA-Yang1-BR, was obtained with a brain metastatic rate of 50%. The use of MRI for the detection of brain metastases has obvious advantages. An experimental Chinese lung adenocarcinoma cell clone (CPA-Yang1-BR) and its animal model with brain metastasis potency in nude mice were established. MRI with sense body or micro MRI may be used as a sensitive, accurate, and noninvasive method to detect experimental brain metastases in intact live immunodeficient mice. The results of this study may serve as a technical platform for brain metastases from lung adenocarcinoma.

  19. Cultured meat from stem cells: challenges and prospects.

    Science.gov (United States)

    Post, Mark J

    2012-11-01

    As one of the alternatives for livestock meat production, in vitro culturing of meat is currently studied. The generation of bio-artificial muscles from satellite cells has been ongoing for about 15 years, but has never been used for generation of meat, while it already is a great source of animal protein. In order to serve as a credible alternative to livestock meat, lab or factory grown meat should be efficiently produced and should mimic meat in all of its physical sensations, such as visual appearance, smell, texture and of course, taste. This is a formidable challenge even though all the technologies to create skeletal muscle and fat tissue have been developed and tested. The efficient culture of meat will primarily depend on culture conditions such as the source of medium and its composition. Protein synthesis by cultured skeletal muscle cells should further be maximized by finding the optimal combination of biochemical and physical conditions for the cells. Many of these variables are known, but their interactions are numerous and need to be mapped. This involves a systematic, if not systems, approach. Given the urgency of the problems that the meat industry is facing, this endeavor is worth undertaking. As an additional benefit, culturing meat may provide opportunities for production of novel and healthier products. Copyright © 2012 Elsevier Ltd. All rights reserved.

  20. Demethoxycurcumin Retards Cell Growth and Induces Apoptosis in Human Brain Malignant Glioma GBM 8401 Cells

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    Tzuu-Yuan Huang

    2012-01-01

    Full Text Available Demethoxycurcumin (DMC; a curcumin-related demethoxy compound has been recently shown to display antioxidant and antitumor activities. It has also produced a potent chemopreventive action against cancer. In the present study, the antiproliferation (using the MTT assay, DMC was found to have cytotoxic activities against GBM 8401 cell with IC50 values at 22.71 μM and induced apoptosis effects of DMC have been investigated in human brain malignant glioma GBM 8401 cells. We have studied the mitochondrial membrane potential (MMP, DNA fragmentation, caspase activation, and NF-κB transcriptional factor activity. By these approaches, our results indicated that DMC has produced an inhibition of cell proliferation as well as the activation of apoptosis in GBM 8401 cells. Both effects were observed to increase in proportion with the dosage of DMC treatment, and the apoptosis was induced by DMC in human brain malignant glioma GBM 8401 cells via mitochondria- and caspase-dependent pathways.

  1. Oxidative Stress Induces Senescence in Cultured RPE Cells.

    Science.gov (United States)

    Aryan, Nona; Betts-Obregon, Brandi S; Perry, George; Tsin, Andrew T

    2016-01-01

    The aim of this research is to determine whether oxidative stress induces cellular senescence in human retinal pigment epithelial cells. Cultured ARPE19 cells were subjected to different concentrations of hydrogen peroxide to induce oxidative stress. Cells were seeded into 24-well plates with hydrogen peroxide added to cell medium and incubated at 37°C + 5% CO2 for a 90-minute period [at 0, 300, 400 and 800 micromolar (MCM) hydrogen peroxide]. The number of viable ARPE19 cells were recorded using the Trypan Blue Dye Exclusion Method and cell senescence was measured by positive staining for senescence-associated beta-galactosidase (SA-beta-Gal) protein. Without hydrogen peroxide treatment, the number of viable ARPE19 cells increased significantly from 50,000 cells/well to 197,000 within 72 hours. Treatment with hydrogen peroxide reduced this level of cell proliferation significantly (to 52,167 cells at 400 MCM; to 49,263 cells at 800 MCM). Meanwhile, cells with a high level of positive senescence-indicator SA-Beta-Gal-positive staining was induced by hydrogen peroxide treatment (from a baseline level of 12% to 80% at 400 MCM and at 800 MCM). Our data suggests that oxidative stress from hydrogen peroxide treatment inhibited ARPE19 cell proliferation and induced cellular senescence.

  2. Human disc cells in monolayer vs 3D culture: cell shape, division and matrix formation

    Directory of Open Access Journals (Sweden)

    Hanley Edward N

    2000-10-01

    Full Text Available Abstract Background The relationship between cell shape, proliferation, and extracellular matrix (ECM production, important aspects of cell behavior, is examined in a little-studied cell type, the human annulus cell from the intervertebral disc, during monolayer vs three-dimensional (3D culture. Results Three experimental studies showed that cells respond specifically to culture microenvironments by changes in cell shape, mitosis and ECM production: 1 Cell passages showed extensive immunohistochemical evidence of Type I and II collagens only in 3D culture. Chondroitin sulfate and keratan sulfate were abundant in both monolayer and 3D cultures. 2 Cells showed significantly greater proliferation in monolayer in the presence of platelet-derived growth factor compared to cells in 3D. 3 Cells on Matrigel™-coated monolayer substrates became rounded and formed nodular colonies, a finding absent during monolayer growth. Conclusions The cell's in vivo interactions with the ECM can regulate shape, gene expression and other cell functions. The shape of the annulus cell changes markedly during life: the young, healthy disc contains spindle shaped cells and abundant collagen. With aging and degeneration, many cells assume a strikingly different appearance, become rounded and are surrounded by unusual accumulations of ECM products. In vitro manipulation of disc cells provides an experimental window for testing how disc cells from given individuals respond when they are grown in environments which direct cells to have either spindle- or rounded-shapes. In vitro assessment of the response of such cells to platelet-derived growth factor and to Matrigel™ showed a continued influence of cell shape even in the presence of a growth factor stimulus. These findings contribute new information to the important issue of the influence of cell shape on cell behavior.

  3. Brain Cell Swelling During Hypocapnia Increases with Hyperglycemia or Ketosis

    Science.gov (United States)

    Glaser, Nicole; Bundros, Angeliki; Anderson, Steve; Tancredi, Daniel; Lo, Weei; Orgain, Myra; O'Donnell, Martha

    2014-01-01

    Severe hypocapnia increases the risk of DKA-related cerebral injury in children, but the reason for this association is unclear. To determine whether the effects of hypocapnia on the brain are altered during hyperglycemia or ketosis, we induced hypocapnia (pCO2 20 ± 3 mmHg) via mechanical ventilation in three groups of juvenile rats: 25 controls, 22 hyperglycemic rats (serum glucose 451± 78 mg/dL) and 15 ketotic rats (beta-hydroxy butyrate 3.0 ± 1.0 mmol/L). We used magnetic resonance imaging to measure cerebral blood flow (CBF) and apparent diffusion coefficient (ADC) values in these groups and in 17 ventilated rats with normal pCO2 (40±3 mmHg). In a subset (n=35), after 2 hrs of hypocapnia, pCO2 levels were normalized (40±3 mmHg) and ADC and CBF measurements repeated. Declines in CBF with hypocapnia occurred in all groups. Normalization of pCO2 after hypocapnia resulted in striatal hyperemia. These effects were not substantially altered by hyperglycemia or ketosis, however, declines in ADC during hypocapnia were greater during both hyperglycemia and ketosis. We conclude that brain cell swelling associated with hypocapnia is increased by both hyperglycemia and ketosis, suggesting that these metabolic conditions may make the brain more vulnerable to injury during hypocapnia. PMID:24443981

  4. Apple derived cellulose scaffolds for 3D mammalian cell culture.

    Directory of Open Access Journals (Sweden)

    Daniel J Modulevsky

    Full Text Available There are numerous approaches for producing natural and synthetic 3D scaffolds that support the proliferation of mammalian cells. 3D scaffolds better represent the natural cellular microenvironment and have many potential applications in vitro and in vivo. Here, we demonstrate that 3D cellulose scaffolds produced by decellularizing apple hypanthium tissue can be employed for in vitro 3D culture of NIH3T3 fibroblasts, mouse C2C12 muscle myoblasts and human HeLa epithelial cells. We show that these cells can adhere, invade and proliferate in the cellulose scaffolds. In addition, biochemical functionalization or chemical cross-linking can be employed to control the surface biochemistry and/or mechanical properties of the scaffold. The cells retain high viability even after 12 continuous weeks of culture and can achieve cell densities comparable with other natural and synthetic scaffold materials. Apple derived cellulose scaffolds are easily produced, inexpensive and originate from a renewable source. Taken together, these results demonstrate that naturally derived cellulose scaffolds offer a complementary approach to existing techniques for the in vitro culture of mammalian cells in a 3D environment.

  5. A glucose fuel cell for implantable brain-machine interfaces.

    Directory of Open Access Journals (Sweden)

    Benjamin I Rapoport

    Full Text Available We have developed an implantable fuel cell that generates power through glucose oxidation, producing 3.4 μW cm(-2 steady-state power and up to 180 μW cm(-2 peak power. The fuel cell is manufactured using a novel approach, employing semiconductor fabrication techniques, and is therefore well suited for manufacture together with integrated circuits on a single silicon wafer. Thus, it can help enable implantable microelectronic systems with long-lifetime power sources that harvest energy from their surrounds. The fuel reactions are mediated by robust, solid state catalysts. Glucose is oxidized at the nanostructured surface of an activated platinum anode. Oxygen is reduced to water at the surface of a self-assembled network of single-walled carbon nanotubes, embedded in a Nafion film that forms the cathode and is exposed to the biological environment. The catalytic electrodes are separated by a Nafion membrane. The availability of fuel cell reactants, oxygen and glucose, only as a mixture in the physiologic environment, has traditionally posed a design challenge: Net current production requires oxidation and reduction to occur separately and selectively at the anode and cathode, respectively, to prevent electrochemical short circuits. Our fuel cell is configured in a half-open geometry that shields the anode while exposing the cathode, resulting in an oxygen gradient that strongly favors oxygen reduction at the cathode. Glucose reaches the shielded anode by diffusing through the nanotube mesh, which does not catalyze glucose oxidation, and the Nafion layers, which are permeable to small neutral and cationic species. We demonstrate computationally that the natural recirculation of cerebrospinal fluid around the human brain theoretically permits glucose energy harvesting at a rate on the order of at least 1 mW with no adverse physiologic effects. Low-power brain-machine interfaces can thus potentially benefit from having their implanted units

  6. T cell resistance to activation by dendritic cells requires long-term culture in simulated microgravity

    Science.gov (United States)

    Bradley, Jillian H.; Stein, Rachel; Randolph, Brad; Molina, Emily; Arnold, Jennifer P.; Gregg, Randal K.

    2017-11-01

    Immune impairment mediated by microgravity threatens the success of space exploration requiring long-duration spaceflight. The cells of most concern, T lymphocytes, coordinate the host response against microbial and cancerous challenges leading to elimination and long-term protection. T cells are activated upon recognition of specific microbial peptides bound on the surface of antigen presenting cells, such as dendritic cells (DC). Subsequently, this engagement results in T cell proliferation and differentiation into effector T cells driven by autocrine interleukin-2 (IL-2) and other cytokines. Finally, the effector T cells acquire the weaponry needed to destroy microbial invaders and tumors. Studies conducted on T cells during spaceflight, or using Earth-based culture systems, have shown reduced production of cytokines, proliferation and effector functions as compared to controls. This may account for the cases of viral reactivation events and opportunistic infections associated with astronauts of numerous missions. This work has largely been based upon the outcome of T cell activation by stimulatory factors that target select T cell signaling pathways rather than the complex, signaling events related to the natural process of antigen presentation by DC. This study tested the response of an ovalbumin peptide-specific T cell line, OT-II TCH, to activation by DC when the T cells were cultured 24-120 h in a simulated microgravity (SMG) environment generated by a rotary cell culture system. Following 72 h culture of T cells in SMG (SMG-T) or control static (Static-T) conditions, IL-2 production by the T cells was reduced in SMG-T cells compared to Static-T cells upon stimulation by phorbol 12-myristate 13-acetate (PMA) and ionomycin. However, when the SMG-T cells were stimulated with DC and peptide, IL-2 was significantly increased compared to Static-T cells. Such enhanced IL-2 production by SMG-T cells peaked at 72 h SMG culture time and decreased thereafter. When

  7. T cell resistance to activation by dendritic cells requires long-term culture in simulated microgravity.

    Science.gov (United States)

    Bradley, Jillian H; Stein, Rachel; Randolph, Brad; Molina, Emily; Arnold, Jennifer P; Gregg, Randal K

    2017-11-01

    Immune impairment mediated by microgravity threatens the success of space exploration requiring long-duration spaceflight. The cells of most concern, T lymphocytes, coordinate the host response against microbial and cancerous challenges leading to elimination and long-term protection. T cells are activated upon recognition of specific microbial peptides bound on the surface of antigen presenting cells, such as dendritic cells (DC). Subsequently, this engagement results in T cell proliferation and differentiation into effector T cells driven by autocrine interleukin-2 (IL-2) and other cytokines. Finally, the effector T cells acquire the weaponry needed to destroy microbial invaders and tumors. Studies conducted on T cells during spaceflight, or using Earth-based culture systems, have shown reduced production of cytokines, proliferation and effector functions as compared to controls. This may account for the cases of viral reactivation events and opportunistic infections associated with astronauts of numerous missions. This work has largely been based upon the outcome of T cell activation by stimulatory factors that target select T cell signaling pathways rather than the complex, signaling events related to the natural process of antigen presentation by DC. This study tested the response of an ovalbumin peptide-specific T cell line, OT-II TCH, to activation by DC when the T cells were cultured 24-120 h in a simulated microgravity (SMG) environment generated by a rotary cell culture system. Following 72 h culture of T cells in SMG (SMG-T) or control static (Static-T) conditions, IL-2 production by the T cells was reduced in SMG-T cells compared to Static-T cells upon stimulation by phorbol 12-myristate 13-acetate (PMA) and ionomycin. However, when the SMG-T cells were stimulated with DC and peptide, IL-2 was significantly increased compared to Static-T cells. Such enhanced IL-2 production by SMG-T cells peaked at 72 h SMG culture time and decreased thereafter

  8. 3D brain Organoids derived from pluripotent stem cells: promising experimental models for brain development and neurodegenerative disorders.

    Science.gov (United States)

    Lee, Chun-Ting; Bendriem, Raphael M; Wu, Wells W; Shen, Rong-Fong

    2017-08-20

    Three-dimensional (3D) brain organoids derived from human pluripotent stem cells (hPSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), appear to recapitulate the brain's 3D cytoarchitectural arrangement and provide new opportunities to explore disease pathogenesis in the human brain. Human iPSC (hiPSC) reprogramming methods, combined with 3D brain organoid tools, may allow patient-derived organoids to serve as a preclinical platform to bridge the translational gap between animal models and human clinical trials. Studies using patient-derived brain organoids have already revealed novel insights into molecular and genetic mechanisms of certain complex human neurological disorders such as microcephaly, autism, and Alzheimer's disease. Furthermore, the combination of hiPSC technology and small-molecule high-throughput screening (HTS) facilitates the development of novel pharmacotherapeutic strategies, while transcriptome sequencing enables the transcriptional profiling of patient-derived brain organoids. Finally, the addition of CRISPR/Cas9 genome editing provides incredible potential for personalized cell replacement therapy with genetically corrected hiPSCs. This review describes the history and current state of 3D brain organoid differentiation strategies, a survey of applications of organoids towards studies of neurodevelopmental and neurodegenerative disorders, and the challenges associated with their use as in vitro models of neurological disorders.

  9. The effect of hydroxylated PCBs on DCF-fluorescence and cell death in cultured rat cerebellar granule cells

    Energy Technology Data Exchange (ETDEWEB)

    Fonnum, F.; Dreiem, A.; Rykken, S. [Norwegian Defence Research Establishment (Norway); Lehmler, H.Y.; Robertson, L. [Univ. of Iowa (United States); Mariussen, E. [Norwegian Inst. for Air Research (Norway)

    2004-09-15

    We have previously investigated the effects of polychlorinated biphenyls (PCBs) on free radical formation and cell death in cerebellar granule cells. PCBs may be metabolised to hydroxylated polychlorinated biphenyls (HO-PCBs) in the body. Therefore, we wanted to expand our study to include also the HO-PCBs. After hydroxylation, many of the PCBs are conjugated to either glucoronic acid or sulphate, which facilitates their excretion. Nevertheless, some of the HO-PCBs are retained in the body. The structures of some of the HO-PCBs show a certain similarity to the structure of the cathecholamines, and PCBs have been shown to affect the level of cathecholamines in the brain. Therefore, we compared the effect of some HO-PCBs with the effects of PCBs on some important physiological parameters in the brain. In the present communication we have compared the effects of PCB and HO-PCB on formation of DCF-fluorescence, which is used as a measure of reactive oxygen species (ROS) formation, and cell death in cultured rat cerebellar granule cells.

  10. Histological Architecture Underlying Brain-Immune Cell-Cell Interactions and the Cerebral Response to Systemic Inflammation.

    Science.gov (United States)

    Shimada, Atsuyoshi; Hasegawa-Ishii, Sanae

    2017-01-01

    Although the brain is now known to actively interact with the immune system under non-inflammatory conditions, the site of cell-cell interactions between brain parenchymal cells and immune cells has been an open question until recently. Studies by our and other groups have indicated that brain structures such as the leptomeninges, choroid plexus stroma and epithelium, attachments of choroid plexus, vascular endothelial cells, cells of the perivascular space, circumventricular organs, and astrocytic endfeet construct the histological architecture that provides a location for intercellular interactions between bone marrow-derived myeloid lineage cells and brain parenchymal cells under non-inflammatory conditions. This architecture also functions as the interface between the brain and the immune system, through which systemic inflammation-induced molecular events can be relayed to the brain parenchyma at early stages of systemic inflammation during which the blood-brain barrier is relatively preserved. Although brain microglia are well known to be activated by systemic inflammation, the mechanism by which systemic inflammatory challenge and microglial activation are connected has not been well documented. Perturbed brain-immune interaction underlies a wide variety of neurological and psychiatric disorders including ischemic brain injury, status epilepticus, repeated social defeat, and neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. Proinflammatory status associated with cytokine imbalance is involved in autism spectrum disorders, schizophrenia, and depression. In this article, we propose a mechanism connecting systemic inflammation, brain-immune interface cells, and brain parenchymal cells and discuss the relevance of basic studies of the mechanism to neurological disorders with a special emphasis on sepsis-associated encephalopathy and preterm brain injury.

  11. Mesenchymal stem cells enhance the metastasis of 3D-cultured hepatocellular carcinoma cells

    International Nuclear Information System (INIS)

    Liu, Chang; Liu, Yang; Xu, Xiao-xi; Guo, Xin; Sun, Guang-wei; Ma, Xiao-jun

    2016-01-01

    Accumulating evidences have demonstrated that mesenchymal stem cells (MSC) could be recruited to the tumor microenvironment. Umbilical cord mesenchymal stem cells (UCMSC) were attractive vehicles for delivering therapeutic agents against cancer. Nevertheless, the safety of UCMSC in the treatment of tumors including hepatocellular carcinoma (HCC) was still undetermined. In this study, an in vitro co-culture system was established to evaluate the effect of UCMSC on the cell growth, cancer stem cell (CSC) characteristics, drug resistance, metastasis of 3D-cultured HCC cells, and the underlying mechanism was also investigated. It was found that after co-cultured with UCMSC, the metastatic ability of 3D-cultured HCC cells was significantly enhanced as indicated by up-regulation of matrix metalloproteinase (MMP), epithelial-mesenchymal transition (EMT)-related genes, and migration ability. However, cell growth, drug resistance and CSC-related gene expression of HCC cells were not affected by UCMSC. Moreover, EMT was reversed, MMP-2 expression was down-regulated, and migration ability of HCC cell was significantly inhibited when TGF-β receptor inhibitor SB431542 was added into the co-culture system. Therefore, these data indicated that UCMSC could significantly enhance the tumor cell metastasis, which was due to the EMT of HCC cells induced by TGF-β. The online version of this article (doi:10.1186/s12885-016-2595-4) contains supplementary material, which is available to authorized users

  12. In vitro plant regeneration from embryogenic cell suspension culture ...

    African Journals Online (AJOL)

    STORAGESEVER

    2008-05-02

    May 2, 2008 ... plasts in A. adsurgens (Luo and Jia, 1998a, b). There are only few reports available on plant regeneration systems via somatic embryogenesis (Luo et al., 1999; Hou and. Jia, 2004). Here, we report a protocol for plant regenera- tion from embryogenic cell suspension culture of endemic. A. chrysochlorus.

  13. Spontaneous calcium waves in granule cells in cerebellar slice cultures

    DEFF Research Database (Denmark)

    Apuschkin, Mia; Ougaard, Maria; Rekling, Jens C

    2013-01-01

    with MK-801. Whole-cell recordings during wave formation showed cyclic EPSP barrages with an amplitude of 10-20 mV concurrent with wave activity. Local non-propagating putative transglial waves were also present in the cultures, and could be reproduced by pressure application of ATP. We hypothesize...

  14. Plant Cell Cultures as Source of Cosmetic Active Ingredients

    Directory of Open Access Journals (Sweden)

    Ani Barbulova

    2014-04-01

    Full Text Available The last decades witnessed a great demand of natural remedies. As a result, medicinal plants have been increasingly cultivated on a commercial scale, but the yield, the productive quality and the safety have not always been satisfactory. Plant cell cultures provide useful alternatives for the production of active ingredients for biomedical and cosmetic uses, since they represent standardized, contaminant-free and biosustainable systems, which allow the production of desired compounds on an industrial scale. Moreover, thanks to their totipotency, plant cells grown as liquid suspension cultures can be used as “biofactories” for the production of commercially interesting secondary metabolites, which are in many cases synthesized in low amounts in plant tissues and differentially distributed in the plant organs, such as roots, leaves, flowers or fruits. Although it is very widespread in the pharmaceutical industry, plant cell culture technology is not yet very common in the cosmetic field. The aim of the present review is to focus on the successful research accomplishments in the development of plant cell cultures for the production of active ingredients for cosmetic applications.

  15. Establishment of the callus and cell suspension culture of ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-10-05

    Oct 5, 2009 ... The objective of this work was the optimization of the conditions of callus and cell suspension culture of Elaeagnus angustifolia for the production of condensed tannins. The effects of different conditions on the callus growth and the production of condensed tannins were researched. The leaf tissue part of.

  16. Establishment of sorghum cell suspension culture system for ...

    African Journals Online (AJOL)

    STORAGESEVER

    2008-03-18

    Mar 18, 2008 ... Key words: Sorghum, proteomics, callus, cell suspension cultures, total soluble protein, secretome. INTRODUCTION. Sorghum, a cereal crop native to Africa, is drought- tolerant, surviving periods of water deficit (Rosenow et al., 1983). The crop is grown in the semi-arid regions of. Africa and Asia primarily ...

  17. Test chambers for cell culture in static magnetic field

    Energy Technology Data Exchange (ETDEWEB)

    Glinka, Marek, E-mail: mag@iq.pl [Research and Development Centre of Electrical Machines. 188 Rozdzienskiego Street, 40-203 Katowice (Poland); Gawron, Stanisław, E-mail: s.gawron@komel.katowice.pl [Research and Development Centre of Electrical Machines. 188 Rozdzienskiego Street, 40-203 Katowice (Poland); Sieroń, Aleksander, E-mail: sieron1@tlen.pl [Department of Internal Diseases, Angiology and Physical Medicine in Bytom. Medical University of Silesia in Katowice. 15 Batorego Street, 41-902 Bytom (Poland); Pawłowska–Góral, Katarzyna, E-mail: kgoral@sum.edu.pl [Department of Food and Nutrition in Sosnowiec. Medical University of Silesia in Katowice. 8 Jednosci Street, 41-200 Sosnowiec (Poland); Cieślar, Grzegorz, E-mail: cieslar1@tlen.pl [Department of Internal Diseases, Angiology and Physical Medicine in Bytom. Medical University of Silesia in Katowice. 15 Batorego Street, 41-902 Bytom (Poland); Sieroń–Stołtny, Karolina [Department of Internal Diseases, Angiology and Physical Medicine in Bytom. Medical University of Silesia in Katowice. 15 Batorego Street, 41-902 Bytom (Poland)

    2013-04-15

    Article presents a test chamber intended to be used for in vitro cell culture in homogenous constant magnetic field with parametrically variable magnitude. We constructed test chambers with constant parameters of control homeostasis of cell culture for the different parameters of static magnetic field. The next step was the computer calculation of 2D and 3D simulation of the static magnetic field distribution in the chamber. The analysis of 2D and 3D calculations of magnetic induction in the cells' exposition plane reveals, in comparison to the detection results, the greater accuracy of 2D calculations (Figs. 9 and 10). The divergence in 2D method was 2–4% and 8 to 10% in 3D method (reaching 10% only out of the cells′ cultures margins). -- Highlights: ► We present test chamber to be used for in vitro cell culture in static magnetic field. ► The technical data of the chamber construction was presented. ► 2D versus 3D simulation of static magnetic field distribution in chamber was reported. ► We report the accuracy of 2D calculation than 3D.

  18. Establishment of the callus and cell suspension culture of ...

    African Journals Online (AJOL)

    The objective of this work was the optimization of the conditions of callus and cell suspension culture of Elaeagnus angustifolia for the production of condensed tannins. The effects of different conditions on the callus growth and the production of condensed tannins were researched. The leaf tissue part of E. angustifolia was ...

  19. Chloride secretion by cultures of pig tracheal gland cells

    Science.gov (United States)

    Borthwell, Rachel M.; Hajighasemi-Ossareh, Mohammad; Lachowicz-Scroggins, Marrah E.; Finkbeiner, W. E.; Stevens, Jeremy E.; Modlin, Sara

    2012-01-01

    Malfunction of airway submucosal glands contributes to the pathology of cystic fibrosis (CF), and cell cultures of CF human airway glands show defects in Cl− and water transport. Recently, a transgenic pig model of CF (the CF pig) has been developed. Accordingly, we have developed cell cultures of pig airway gland epithelium for use in investigating alterations in gland function in CF. Our cultures form tight junctions (as evidenced by high transepithelial electrical resistance) and show high levels of active anion secretion (measured as amiloride-insensitive short-circuit current). In agreement with recent results on human airway glands, neurohumoral agents that elevate intracellular Ca2+ potently stimulated anion secretion, while elevation of cAMP was comparatively ineffective. Our cultures express lactoferrin and lysozyme (serous gland cell markers) and MUC5B (the main mucin of airway glands). They are, therefore, potentially useful in determining if CF-related alterations in anion transport result in altered secretion of serous cell antimicrobial agents or mucus. PMID:22367783

  20. [Colorectal cancer: tissutal explantation and primary cell culture].

    Science.gov (United States)

    Spisni, Roberto; Failli, Alessandra; Orsini, Giulia; Kastsiuchenka, Olga; Natale, Gianfranco; Castagna, Maura; Legitimo, Annalisa; Aghasbabyan, Alekandr; Ambrosini, Carlo Enrico; Consolini, Rita; Miccoli, Paolo

    2009-01-01

    Setting of cellular cultures extracted from colorectal cancer tissue represents a valid model for in vitro study of biological and molecular characteristics of each single tumor finalized to obtain a tailored chemiotherapy. The end point of this study is to create primary cellular cultures from "fresh" cancer tissue in different stages of evolution. Cancer tissue samples are obtained by means of surgical excisional biopsy or by means of semi-automatic biopsy instrument (Sprig-Cut). After having compared different approaches, two experimental protocols have been selected to have the highest number or intact cells: enzimatic digestion with trypsin and explantation. Primary cell culture free of microbic contamination, obtained mainly by means of Spring-Cut methods, underwent immunohistochemical analysis to evaluate what kind of cell have been grown in vitro by measuring the expression of CK20 and GFAP both resulted positive. The possibility of setting a primary cell culture which represents the cancer of each patient allows a pharmacologic and biomolecular study which can contribute to the development of a tailored adjuvant therapy with many advantages for the patient in terms of positive answer to the treatment and reduced toxicity.

  1. Disposable Bioreactors for Plant Micropropagation and Mass Plant Cell Culture

    Science.gov (United States)

    Ducos, Jean-Paul; Terrier, Bénédicte; Courtois, Didier

    Different types of bioreactors are used at Nestlé R&D Centre - Tours for mass propagation of selected plant varieties by somatic embryogenesis and for large scale culture of plants cells to produce metabolites or recombinant proteins. Recent studies have been directed to cut down the production costs of these two processes by developing disposable cell culture systems. Vegetative propagation of elite plant varieties is achieved through somatic embryogenesis in liquid medium. A pilot scale process has recently been set up for the industrial propagation of Coffea canephora (Robusta coffee). The current production capacity is 3.0 million embryos per year. The pre-germination of the embryos was previously conducted by temporary immersion in liquid medium in 10-L glass bioreactors. An improved process has been developed using a 10-L disposable bioreactor consisting of a bag containing a rigid plastic box ('Box-in-Bag' bioreactor), insuring, amongst other advantages, a higher light transmittance to the biomass due to its horizontal design. For large scale cell culture, two novel flexible plastic-based disposable bioreactors have been developed from 10 to 100 L working volumes, validated with several plant species ('Wave and Undertow' and 'Slug Bubble' bioreactors). The advantages and the limits of these new types of bioreactor are discussed, based mainly on our own experience on coffee somatic embryogenesis and mass cell culture of soya and tobacco.

  2. CYTOTOXICITY TESTING OF WOUND DRESSINGS USING METHYLCELLULOSE CELL-CULTURE

    NARCIS (Netherlands)

    VANLUYN, MJA; VANWACHEM, PB; NIEUWENHUIS, P; JONKMAN, MF

    1992-01-01

    Wound dressings may induce cytotoxic effects. In this study, we check several, mostly commercially available, wound dressings for cytotoxicity. We used our previously described, newly developed and highly sensitive 7 d methylcellulose cell culture with fibroblasts as the test system. Cytotoxicity is

  3. Lethal impacts of cigarette smoke in cultured tobacco cells

    Directory of Open Access Journals (Sweden)

    Kawano Tomonori

    2011-07-01

    Full Text Available Abstract Background In order to understand and generalize the toxic mechanism of cigarette smoke in living cells, comparison of the data between animal systems and other biological system such as microbial and plant systems is highly beneficial. Objective By employing the tobacco cells as model materials for cigarette smoke toxicity assay, the impacts of the combustion by-products such as nitrogen oxides could be highlighted as the toxic impacts of the plant-derived endogenous chemicals could be excluded in the plant cells. Methods Cigarette smoke-induced cell death was assessed in tobacco cell suspension cultures in the presence and absence of pharmacological inhibitors. Results Cigarette smoke was effective in induction of cell death. The smoke-induced cell death could be partially prevented by addition of nitric oxide (NO scavenger, suggesting the role for NO as the cell death mediator. Addition of NO donor to tobacco cells also resulted in development of partial cell death further confirming the role of NO as cell death mediator. Members of reactive oxygen species and calcium ion were shown to be protecting the cells from the toxic action of smoke-derived NO.

  4. Effect of transplants of retinal pigment epithelial cells from adult human eye on degenerative processes in the brain of rats with experimental acute hypoxia.

    Science.gov (United States)

    Aleksandrova, M A; Kuznetsova, A V; Verdiev, B I; Milyushina-Rzhanova, L A; Sukhinich, K K

    2014-05-01

    Stimulation of cell regeneration in the brain and eye retina in various degenerative processes is a pressing problem in neurobiology. A promising approach is transplantation of somatic cells reprogrammed towards neural lineage. We studied the effect of transplantation of retinal pigment epithelial cells from adult human eye transdifferentiated in culture on degenerative processes in the brain of rats subjected to acute hypoxia. Immunohistochemical and molecular genetic analysis suggests that retinal pigment epithelial cells transdifferentiate in vitro and express markers of low-differentiated neural cells. The cells transplanted into rat brain survive for at least 20 days. During this period, they stimulate compensatory and reparative processes that protected cortical neurons in the recipients from hypoxia-induced degeneration.

  5. Mefloquine damage vestibular hair cells in organotypic cultures.

    Science.gov (United States)

    Yu, Dongzhen; Ding, Dalian; Jiang, Haiyan; Stolzberg, Daniel; Salvi, Richard

    2011-07-01

    Mefloquine is an effective and widely used anti-malarial drug; however, some clinical reports suggest that it can cause dizziness, balance, and vestibular disturbances. To determine if mefloquine might be toxic to the vestibular system, we applied mefloquine to organotypic cultures of the macula of the utricle from postnatal day 3 rats. The macula of the utricle was micro-dissected out as a flat surface preparation and cultured with 10, 50, 100, or 200 μM mefloquine for 24 h. Specimens were stained with TRITC-conjugated phalloidin to label the actin in hair cell stereocilia and TO-PRO-3 to visualize cell nuclei. Some utricles were also labeled with fluorogenic caspase-3, -8, or -9 indicators to evaluate the mechanism of programmed cell death. Mefloquine treatment caused a dose-dependent loss of utricular hair cells. Treatment with 10 μM caused a slight reduction, 50 μM caused a significant reduction, and 200 μM destroyed nearly all the hair cells. Hair cell nuclei in mefloquine-treated utricles were condensed and fragmented, morphological features of apoptosis. Mefloquine-treated utricles were positive for the extrinsic initiator caspase-8 and intrinsic initiator caspase-9 and downstream executioner caspase-3. These results indicate that mefloquine can induce significant hair cell degeneration in the postnatal rat utricle and that mefloquine-induced hair cell death is initiated by both caspase-8 and caspase-9.

  6. Ultrastructure of cells of Ulmus americana cultured in vitro and exposed to the culture filtrate of Ceratocystis ulmi

    Science.gov (United States)

    Paula M. Pijut; R. Daniel Lineberger; Subhash C. Domir; Jann M. Ichida; Charles R. Krause

    1990-01-01

    Calli of American elm susceptible and resistant to Dutch elm disease were exposed to a culture filtrate of a pathogenic isolate of Ceratocystis ulmi. Cells from untreated tissue exhibited typical internal composition associated with healthy, actively growing cells. All cells exposed to culture filtrate showed appreciable ultrastructural changes....

  7. Brain Region-Dependent Rejection of Neural Precursor Cell Transplants

    Directory of Open Access Journals (Sweden)

    Nina Fainstein

    2018-04-01

    Full Text Available The concept of CNS as an immune-privileged site has been challenged by the occurrence of immune surveillance and allogeneic graft rejection in the brain. Here we examined whether the immune response to allogeneic neural grafts is determined by the site of implantation in the CNS. Dramatic regional differences were observed between immune responses to allogeneic neural precursor/stem cell (NPC grafts in the striatum vs. the hippocampus. Striatal grafts were heavily infiltrated with IBA-1+ microglia/macrophages and CD3+ T cells and completely rejected. In contrast, hippocampal grafts exhibited milder IBA-1+ cell infiltration, were not penetrated efficiently by CD3+ cells, and survived efficiently for at least 2 months. To evaluate whether the hippocampal protective effect is universal, astrocytes were then transplanted. Allogeneic astrocyte grafts elicited a vigorous rejection process from the hippocampus. CD200, a major immune-inhibitory signal, plays an important role in protecting grafts from rejection. Indeed, CD200 knock out NPC grafts were rejected more efficiently than wild type NPCs from the striatum. However, lack of CD200 expression did not elicit NPC graft rejection from the hippocampus. In conclusion, the hippocampus has partial immune-privilege properties that are restricted to NPCs and are CD200-independent. The unique hippocampal milieu may be protective for allogeneic NPC grafts, through host-graft interactions enabling sustained immune-regulatory properties of transplanted NPCs. These findings have implications for providing adequate immunosuppression in clinical translation of cell therapy.

  8. Nootropic agents stimulate neurogenesis. Brain Cells, Inc.: WO2007104035.

    Science.gov (United States)

    Taupin, Philippe

    2009-05-01

    The application is in the field of adult neurogenesis, neural stem cells and cellular therapy. It aims to characterize the activity of nootropic agents on adult neurogenesis in vitro. Nootropic agents are substances improving cognitive and mental abilities. AMPA (alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate) and nootropic agents were assessed for the potential to differentiate human neural progenitor and stem cells into neuronal cells in vitro. They were also tested for their behavioural activity on the novel object recognition task. AMPA, piracetam, FK-960 and SGS-111 induce and stimulate neuronal differentiation of human-derived neural progenitor and stem cells. SGS-111 increases the number of visits to the novel object. The neurogenic activity of piracetam and SGS-111 is mediated through AMPA receptor. The neurogenic activity of SGS-111 may contribute and play a role in its nootropic activity. These results suggest that nootropic agents may elicit some of their effects through their neurogenic activity. The application claims the use of nootropic agents for their neurogenic activity and for the treatment of neurological diseases, disorders and injuries, by stimulating or increasing the generation of neuronal cells in the adult brain.

  9. Differential heat shock response of primary human cell cultures and established cell lines

    DEFF Research Database (Denmark)

    Richter, W W; Issinger, O G

    1986-01-01

    degrees C treatment, whereas in immortalized cell lines usually 90% of the cells were found in suspension. Enhanced expression of the major heat shock protein (hsp 70) was found in all heat-treated cells. In contrast to the primary cell cultures, established and transformed cell lines synthesized...... a protein with an apparent molecular mass of 70 kDa and an isoelectric pH of 7.0 as early as 3 h after the initial hyperthermal treatment....

  10. Morphological differences between circulating tumor cells from prostate cancer patients and cultured prostate cancer cells.

    Directory of Open Access Journals (Sweden)

    Sunyoung Park

    Full Text Available Circulating tumor cell (CTC enumeration promises to be an important predictor of clinical outcome for a range of cancers. Established CTC enumeration methods primarily rely on affinity capture of cell surface antigens, and have been criticized for underestimation of CTC numbers due to antigenic bias. Emerging CTC capture strategies typically distinguish these cells based on their assumed biomechanical characteristics, which are often validated using cultured cancer cells. In this study, we developed a software tool to investigate the morphological properties of CTCs from patients with castrate resistant prostate cancer and cultured prostate cancer cells in order to establish whether the latter is an appropriate model for the former. We isolated both CTCs and cultured cancer cells from whole blood using the CellSearch® system and examined various cytomorphological characteristics. In contrast with cultured cancer cells, CTCs enriched by CellSearch® system were found to have significantly smaller size, larger nuclear-cytoplasmic ratio, and more elongated shape. These CTCs were also found to exhibit significantly more variability than cultured cancer cells in nuclear-cytoplasmic ratio and shape profile.

  11. Neuropharmacological and neuroprotective activities of some metabolites produced by cell suspension culture of Waltheria americana Linn.

    Science.gov (United States)

    Mundo, Jorge; Villeda-Hernández, Juana; Herrera-Ruiz, Maribel; Gutiérrez, María Del Carmen; Arellano-García, Jesús; León-Rivera, Ismael; Perea-Arango, Irene

    2017-10-01

    Waltheria americana is a plant used in Mexican traditional medicine to treat some nervous system disorders. The aims of the present study were to isolate and determine the neuropharmacological and neurprotective activities of metabolites produced by a cell suspension culture of Waltheria americana. Submerged cultivation of W. americana cells provided biomass. A methanol-soluble extract (WAsc) was obtained from biomass. WAsc was fractionated yielding the chromatographic fractions 4WAsc-H 2 O and WAsc-CH 2 Cl 2 . For the determination of anticonvulsant activity in vivo, seizures were induced in mice by pentylenetetrazol (PTZ). Neuropharmacological activities (release of gamma amino butyric acid (GABA) and neuroprotection) of chromatographic fractions were determined by in vitro histological analysis of brain sections of mice post mortem. Fraction 4WAsc-H 2 O (containing saccharides) did not produce neuronal damage, neurodegeneration, interstitial tissue edema, astrocytic activation, nor cell death. Pretreatment of animals with 4WAsc-H 2 O and WAsc-CH 2 Cl 2 from W. americana cell suspensions induced an increase in: GABA release, seizure latency, survival time, neuroprotection, and a decrease in the degree of severity of tonic/tonic-clonic convulsions, preventing PTZ-induced death of up to 100% of animals of study. Bioactive compounds produced in suspension cell culture of W. americana produce neuroprotective and neuropharmacological activities associated with the GABAergic neurotransmission system. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  12. Rapid Detection of Apoptosis in Cultured Mammalian Cells.

    Science.gov (United States)

    Kudryavtsev, Igor; Serebryakova, Maria; Solovjeva, Liudmila; Svetlova, Maria; Firsanov, Denis

    2017-01-01

    Flow cytometry is a powerful tool for the analysis of apoptosis, the process that directly determines cell fate after the action of different stresses. Here, we describe a flow cytometry method for the assessment of early and late stages of apoptosis in non-fixed cultured cells using SYTO16, DRAQ7, and PO-PRO1 dyes simultaneously. This multicolor flow cytometry procedure requires 45 min for completion and provides a quantitative assessment of cell viability. It can be useful in evaluating the cytotoxic properties of new drugs, and antitumor interventions.

  13. A novel 3D human glioblastoma cell culture system for modeling drug and radiation responses.

    Science.gov (United States)

    Gomez-Roman, Natividad; Stevenson, Katrina; Gilmour, Lesley; Hamilton, Graham; Chalmers, Anthony J

    2017-02-01

    Glioblastoma (GBM) is the most common primary brain tumor, with dismal prognosis. The failure of drug-radiation combinations with promising preclinical data to translate into effective clinical treatments may relate to the use of simplified 2-dimensional in vitro GBM cultures. We developed a customized 3D GBM culture system based on a polystyrene scaffold (Alvetex) that recapitulates key histological features of GBM and compared it with conventional 2D cultures with respect to their response to radiation and to molecular targeted agents for which clinical data are available. In 3 patient-derived GBM lines, no difference in radiation sensitivity was observed between 2D and 3D cultures, as measured by clonogenic survival. Three different molecular targeted agents, for which robust clinical data are available were evaluated in 2D and 3D conditions: (i) temozolomide, which improves overall survival and is standard of care for GBM, exhibited statistically significant effects on clonogenic survival in both patient-derived cell lines when evaluated in the 3D model compared with only one cell line in 2D cells; (ii) bevacizumab, which has been shown to increase progression-free survival when added to standard chemoradiation in phase III clinical trials, exhibited marked radiosensitizing activity in our 3D model but had no effect on 2D cells; and (iii) erlotinib, which had no efficacy in clinical trials, displayed no activity in our 3D GBM model, but radiosensitized 2D cells. Our 3D model reliably predicted clinical efficacy, strongly supporting its clinical relevance and potential value in preclinical evaluation of drug-radiation combinations for GBM. © The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Neuro-Oncology.

  14. Co-cultures with stem cell-derived human sensory neurons reveal regulators of peripheral myelination.

    Science.gov (United States)

    Clark, Alex J; Kaller, Malte S; Galino, Jorge; Willison, Hugh J; Rinaldi, Simon; Bennett, David L H

    2017-04-01

    . Myelinating co-cultures using human induced pluripotent stem cell-derived sensory neurons thus provide insights into the cellular and molecular specialization of axoglial signalling, how pharmacological agents may promote or impede such signalling and the pathogenic effects of ganglioside antibodies.awx012media15372351982001. © The Author (2017). Published by Oxford University Press on behalf of the Guarantors of Brain.

  15. Synchronization of stochastic systems: from paddlefish electroreceptors to human epileptic glial cell cultures

    Science.gov (United States)

    Neiman, Alexander

    2000-03-01

    Synchronization is one of the fundamental nonlinear phenomena observed in nature. We have studied stochastic synchronization in the electrosensitive system of the paddlefish, Polyodon spathula and have also applied synchronization analysis to networks of glial cells cultured from brain tissue of patients with severe epilepsy. We also present theoretical and numerical models for stochastic synchronization. The electrosensitive system of the paddlefish consists of tens of thousands of electroreceptors located mainly on the "rostrum", which serves as an antenna to locate plankton. Each electroreceptor is a noisy oscillator with natural frequencies in the range of 30-90 Hz. We study synchronization in vivo due to 3-20 Hz external periodic electric fields, which correspond to natural signals produced by Daphnia, the usual prey of paddlefish. We find that for signals whose strengths are in the range that paddlefish customarily encounter in the wild, synchronization coding offers a plausible alternative to the more usual rate coding. We also have studied mutual synchronization between different electroreceptors. Although the spontaneous firing of distant electroreceptors is not synchronized, synchronization is observed when external periodic or even noisy electric fields are applied. We have applied the same analysis techniques to examine synchronization between groups of glial cells. In contrast to cultures of healthy astrocytes, which demonstrate calcium waves, the networks from epileptic tissue are characterized by spatially disordered hyper activity. Nevertheless, we have found that, in many cases, synchronized activity is a rather typical for tissue taken from the uncus region of the brain.

  16. Effect of Micro Ridges on Orientation of Cultured Cell

    Directory of Open Access Journals (Sweden)

    Haruka Hino

    2014-06-01

    Full Text Available The effect of micro ridges on orientation of cultured cells has been studied in vitro. Several patterns of micro ridges have been fabricated on a transparent polydimethylsiloxane disk with the photo lithography technique. The ridges consist of several lines of rectangular column: the width of 0.003 mm, the interval of 0.007 mm. Variation has been made on the height of the ridge between 0.0003 mm and 0.0035 mm. C2C12 (mouse myoblast cell line originated with cross-striated muscle of C3H mouse was cultured on the disk with the micro ridges for one week and was observed with an inverted phase contrast microscope. The experimental results show that cells adhere on the top of the ridge and align to the longitudinal direction of the micro ridges with the height between 0.0015 mm and 0.0025 mm.

  17. Improved Cell Culture Method for Growing Contracting Skeletal Muscle Models

    Science.gov (United States)

    Marquette, Michele L.; Sognier, Marguerite A.

    2013-01-01

    An improved method for culturing immature muscle cells (myoblasts) into a mature skeletal muscle overcomes some of the notable limitations of prior culture methods. The development of the method is a major advance in tissue engineering in that, for the first time, a cell-based model spontaneously fuses and differentiates into masses of highly aligned, contracting myotubes. This method enables (1) the construction of improved two-dimensional (monolayer) skeletal muscle test beds; (2) development of contracting three-dimensional tissue models; and (3) improved transplantable tissues for biomedical and regenerative medicine applications. With adaptation, this method also offers potential application for production of other tissue types (i.e., bone and cardiac) from corresponding precursor cells.

  18. Plant cell tissue culture: A potential source of chemicals

    Energy Technology Data Exchange (ETDEWEB)

    Scott, C.D.; Dougall, D.K.

    1987-08-01

    Higher plants produce many industrially important products. Among these are drugs and medicinal chemicals, essential oils and flavors, vegetable oils and fats, fine and specialty chemicals, and even some commodity chemicals. Although, currently, whole-plant extraction is the primary means of harvesting these materials, the advent of plant cell tissue culture could be a much more effective method of producing many types of phytochemicals. The use of immobilized plant cells in an advanced bioreactor configuration with excretion of the product into the reactor medium may represent the most straightforward way of commercializing such techniques for lower-value chemicals. Important research and development opportunities in this area include screening for plant cultures for nonmedical, lower-value chemicals; understanding and controlling plant cell physiology and biochemistry; optimizing effective immobilization methods; developing more efficient bioreactor concepts; and perfecting product extraction and purification techniques. 62 refs., 2 figs.

  19. Fermented Chinese Formula Shuan-Tong-Ling Protects Brain Microvascular Endothelial Cells against Oxidative Stress Injury

    Directory of Open Access Journals (Sweden)

    Lingjing Tan

    2016-01-01

    Full Text Available Fermented Chinese formula Shuan-Tong-Ling (STL, composed of fourteen medicinal herbs, was an experiential formula by Dr. Zhigang Mei for treating vascular encephalopathy, but the underlying mechanisms remained unknown. In this study, we aimed to investigate the protective effects of fermented STL on hydrogen peroxide- (H2O2- induced injury in rat brain microvascular endothelial cells (BMECs and the possible mechanisms. Cultured BMECs were treated with H2O2, STL, or nicotinamide (NAM, a SIRT1 inhibitor. Then, 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyl-2H-tetrazolium bromide (MTT assay was employed to detect cell proliferation and senescence-associated beta-galactosidase (SA-β-gal was used to examine cell senescence. Cell nuclei were observed by 4′,6-diamidino-2-phenylindole. Additionally, changes in reactive oxygen species (ROS, superoxide dismutase (SOD, and glutathione (GSH levels were measured. Expression of SIRT1, p21, and PGC-1α was determined by western blot. Cell proliferation significantly increased with STL treatment in a dose-dependent manner. H2O2 treatment could intensify cell senescence and nuclei splitting or pyknosis. With STL treatment, the reduced ROS level was accompanied by increased SOD and GSH activity. Further assays showed upregulation of SIRT1 and PGC-1α and downregulation of p21 after STL treatment. The results revealed that STL could protect BMECs against oxidative stress injury at least partially through the SIRT1 pathway.

  20. PDMS/glass microfluidic cell culture system for cytotoxicity tests and cells passage

    DEFF Research Database (Denmark)

    Ziolkowska, K.; Jedrych, E.; Kwapiszewski, R.

    2010-01-01

    In this paper, hybrid (PDMS/glass) microfluidic cell culture system (MCCS) integrated with the concentration gradient generator (CGG) is presented. PDMS gas permeability enabled cells' respiration in the fabricated microdevices and excellent glass hydrophilicity allowed successful cells' seeding......' bioactivity, defining the lowest toxic level of tested substances etc....

  1. Adenosine formation in contracting primary rat skeletal muscle cells and endothelial cells in culture

    DEFF Research Database (Denmark)

    Hellsten, Ylva; Frandsen, Ulrik

    1997-01-01

    1. The present study examined the capacity for adenosine formation, uptake and metabolism in contracting primary rat muscle cells and in microvascular endothelial cells in culture. 2. Strong and moderate electrical simulation of skeletal muscle cells led to a significantly greater increase...

  2. Engineering systems for the generation of patterned co-cultures for controlling cell-cell interactions.

    Science.gov (United States)

    Kaji, Hirokazu; Camci-Unal, Gulden; Langer, Robert; Khademhosseini, Ali

    2011-03-01

    Inside the body, cells lie in direct contact or in close proximity to other cell types in a tightly controlled architecture that often regulates the resulting tissue function. Therefore, tissue engineering constructs that aim to reproduce the architecture and the geometry of tissues will benefit from methods of controlling cell-cell interactions with microscale resolution. We discuss the use of microfabrication technologies for generating patterned co-cultures. In addition, we categorize patterned co-culture systems by cell type and discuss the implications of regulating cell-cell interactions in the resulting biological function of the tissues. Patterned co-cultures are a useful tool for fabricating tissue engineered constructs and for studying cell-cell interactions in vitro, because they can be used to control the degree of homotypic and heterotypic cell-cell contact. In addition, this approach can be manipulated to elucidate important factors involved in cell-matrix interactions. Patterned co-culture strategies hold significant potential to develop biomimetic structures for tissue engineering. It is expected that they would create opportunities to develop artificial tissues in the future. This article is part of a Special Issue entitled Nanotechnologies - Emerging Applications in Biomedicine. 2010 Elsevier B.V. All rights reserved.

  3. MEMS-based dynamic cell-to-cell culture platforms using electrochemical surface modifications

    International Nuclear Information System (INIS)

    Chang, Jiyoung; Lin, Liwei; Yoon, Sang-Hee; Mofrad, Mohammad R K

    2011-01-01

    MEMS-based biological platforms with the capability of both spatial placements and time releases of living cells for cell-to-cell culture experiments have been designed and demonstrated utilizing electrochemical surface modification effects. The spatial placement is accomplished by electrochemical surface modification of substrate surfaces to be either adhesive or non-adhesive for living cells. The time control is achieved by the electrical activation of the selective indium tin oxide co-culture electrode to allow the migration of living cells onto the electrode to start the cell-to-cell culture studies. Prototype devices have a three-electrode design with an electrode size of 50 × 50 µm 2 and the separation gaps of 2 µm between them. An electrical voltage of −1.5 V has been used to activate the electrodes independently and sequentially to demonstrate the dynamic cell-to-cell culture experiments of NIH 3T3 fibroblast and Madin Darby canine kidney cells. As such, this MEMS platform could be a basic yet versatile tool to characterize transient cell-to-cell interactions

  4. Cell Migration in Tissues: Explant Culture and Live Imaging.

    Science.gov (United States)

    Staneva, Ralitza; Barbazan, Jorge; Simon, Anthony; Vignjevic, Danijela Matic; Krndija, Denis

    2018-01-01

    Cell migration is a process that ensures correct cell localization and function in development and homeostasis. In disease such as cancer, cells acquire an upregulated migratory capacity that leads to their dissemination throughout the body. Live imaging of cell migration allows for better understanding of cell behaviors in development, adult tissue homeostasis and disease. We have optimized live imaging procedures to track cell migration in adult murine tissue explants derived from: (1) healthy gut; (2) primary intestinal carcinoma; and (3) the liver, a common metastatic site. To track epithelial cell migration in the gut, we generated an inducible fluorescent reporter mouse, enabling us to visualize and track individual cells in unperturbed gut epithelium. To image intratumoral cancer cells, we use a spontaneous intestinal cancer model based on the activation of Notch1 and deletion of p53 in the mouse intestinal epithelium, which gives rise to aggressive carcinoma. Interaction of cancer cells with a metastatic niche, the mouse liver, is addressed using a liver colonization model. In summary, we describe a method for long-term 3D imaging of tissue explants by two-photon excitation microscopy. Explant culturing and imaging can help understand dynamic behavior of cells in homeostasis and disease, and would be applicable to various tissues.

  5. [Neuroscience and collective memory: memory schemas linking brain, societies and cultures].

    Science.gov (United States)

    Legrand, Nicolas; Gagnepain, Pierre; Peschanski, Denis; Eustache, Francis

    2015-01-01

    During the last two decades, the effect of intersubjective relationships on cognition has been an emerging topic in cognitive neurosciences leading through a so-called "social turn" to the formation of new domains integrating society and cultures to this research area. Such inquiry has been recently extended to collective memory studies. Collective memory refers to shared representations that are constitutive of the identity of a group and distributed among all its members connected by a common history. After briefly describing those evolutions in the study of human brain and behaviors, we review recent researches that have brought together cognitive psychology, neuroscience and social sciences into collective memory studies. Using the reemerging concept of memory schema, we propose a theoretical framework allowing to account for collective memories formation with a specific focus on the encoding process of historical events. We suggest that (1) if the concept of schema has been mainly used to describe rather passive framework of knowledge, such structure may also be implied in more active fashions in the understanding of significant collective events. And, (2) if some schema researches have restricted themselves to the individual level of inquiry, we describe a strong coherence between memory and cultural frameworks. Integrating the neural basis and properties of memory schema to collective memory studies may pave the way toward a better understanding of the reciprocal interaction between individual memories and cultural resources such as media or education. © Société de Biologie, 2016.

  6. Transfection of primary brain capillary endothelial cells for protein synthesis and secretion of recombinant erythropoietin: a strategy to enable protein delivery to the brain

    DEFF Research Database (Denmark)

    Burkhart, Annette; Andresen, Thomas Lars; Aigner, Achim

    2017-01-01

    , as turning BCECs into recombinant protein factories by transfection could result in protein secretion further into the brain. The present study aims to investigate the possibility of transfecting primary rat brain endothelial cells (RBECs) for recombinant protein synthesis and secretion...... of the neuroprotective protein erythropoietin (EPO). We previously showed that 4% of RBECs with BBB properties can be transfected without disrupting the BBB integrity in vitro, but it can be questioned whether this is sufficient to enable protein secretion at therapeutic levels. The present study examined various...... transfection vectors, with regard to increasing the transfection efficiency without disrupting the BBB integrity. Lipofectamine 3000™ was the most potent vector compared to polyethylenimine (PEI) and Turbofect. When co-cultured with astrocytes, the genetically modified RBECs secreted recombinant EPO...

  7. Three-dimensional cell culture model utilization in cancer stem cell research.

    Science.gov (United States)

    Bielecka, Zofia F; Maliszewska-Olejniczak, Kamila; Safir, Ilan J; Szczylik, Cezary; Czarnecka, Anna M

    2017-08-01

    Three-dimensional (3D) cell culture models are becoming increasingly popular in contemporary cancer research and drug resistance studies. Recently, scientists have begun incorporating cancer stem cells (CSCs) into 3D models and modifying culture components in order to mimic in vivo conditions better. Currently, the global cell culture market is primarily focused on either 3D cancer cell cultures or stem cell cultures, with less focus on CSCs. This is evident in the low product availability officially indicated for 3D CSC model research. This review discusses the currently available commercial products for CSC 3D culture model research. Additionally, we discuss different culture media and components that result in higher levels of stem cell subpopulations while better recreating the tumor microenvironment. In summary, although progress has been made applying 3D technology to CSC research, this technology could be further utilized and a greater number of 3D kits dedicated specifically to CSCs should be implemented. © 2016 The Authors. Biological Reviews published by John Wiley & Sons Ltd on behalf of Cambridge Philosophical Society.

  8. [The effect of Solcoseryl on in-vitro cultured cells].

    Science.gov (United States)

    Lindner, G; Grosse, G; Lehmann, A

    1977-01-01

    Explants of peripherical nervous system (PNS), skin and ventriculus cordis from chick embryo were cultivated in Maximow chambers and the effect of Solcoseryl, Fa. Solco Basel AG, on some morphological parameters was tested. 1. The growth of tissue cultures is influenced by Solcoseryl in relation to concentration and time of application. The index of area in cultures of PNS and cor increased within the first days. By long time application up to 6 days in vitro the index of area decreased and the index was the same than in controls. Explants of skin showed no essential stimulation of growth. 2. The number of cells per unit of culture in the outgrowth of PNS, cor and skin was different influenced. The density of cells in cultures of PNS and skin decreased (signif. difference). In explants of heart we could not observe a difference between the inside and outside of the outgrowth. An influence of Solcoseryl on the degree of migration is discussed. 3. The area of cell nuclei from heartcells was observed. The area decreased under the influence of Solcoseryl. The difference is significant. 4. The mitotic index of heart cells increased by application of Solcoseryl within the first 2 and 3 days in vitro. 5. The number of nucleoli per nucleus of heart cells under experimental conditions increased significant. It is discussed, Solcoseryl influenced in vitro metabolic processes in suitable systems; stimulation of cell proliferation and migration and rns-synthesis was observed within the first days of cultivation. In-vitro-systems are important objects and they are suitable for tests of pharmaca in vitro.

  9. Viral antigen production in cell cultures on microcarriers Bovine parainfluenza 3 virus and MDBK cells.

    Science.gov (United States)

    Conceição, M M; Tonso, A; Freitas, C B; Pereira, C A

    2007-11-07

    Viral antigens can be obtained from infected mammalian cells cultivated on microcarriers. We have worked out parameters for the production of bovine parainfluenza 3 (PI-3) virus by Mandin-Darby Bovine Kidney (MDBK) cells cultivated on Cytodex 1 microcarriers (MCs) in spinners flasks and bioreactor using fetal bovine serum (FBS) supplemented Eagle minimal essential medium (Eagle-MEM). Medium renewal during the cell culture was shown to be crucial for optimal MCs loading (>90% MCs with confluent cell monolayers) and cell growth (2.5 x 10(6)cells/mL and a micro(x) (h(-1)) 0.05). Since cell cultures performed with lower amount of MCs (1g/L), showed good performances in terms of cell loading, we designed batch experiments with a lower concentration of MCs in view of optimizing the cell growth and virus production. Studies of cell growth with lower concentrations of MCs (0.85 g/L) showed that an increase in the initial cell seeding (from 7 to 40 cells/MC) led to a different kinetic of initial cell growth but to comparable final cell concentrations ((8-10)x10(5)cells/mL at 120 h) and cell loading (210-270 cells/MC). Upon infection with PI-3 virus, cultures showed a decrease in cell growth and MC loading directly related to the multiplicity of infection (moi) used for virus infection. Infected cultures showed also a higher consumption of glucose and production of lactate. The PI-3 virus and PI-3 antigen production among the cultures was not significantly different and attained values ranging from, respectively, 7-9 log(10) TCID(50)/mL and 1.5-2.2 OD. The kinetics of PI-3 virus production showed a sharp increase during the first 24h and those of PI-3 antigen increased after 24h. The differential kinetics of PI-3 virus and PI-3 antigen can be explained by the virus sensitivity to temperature. In view of establishing a protocol of virus production and based on the previous experiments, MDBK cell cultures performed under medium perfusion in a bioreactor of 1.2L were infected

  10. Intracranial Tumor Cell Migration and the Development of Multiple Brain Metastases in Malignant Melanoma

    Directory of Open Access Journals (Sweden)

    Trude G. Simonsen

    2016-06-01

    Full Text Available INTRODUCTION: A majority of patients with melanoma brain metastases develop multiple lesions, and these patients show particularly poor prognosis. To develop improved treatment strategies, detailed insights into the biology of melanoma brain metastases, and particularly the development of multiple lesions, are needed. The purpose of this preclinical investigation was to study melanoma cell migration within the brain after cell injection into a well-defined intracerebral site. METHODS: A-07, D-12, R-18, and U-25 human melanoma cells transfected with green fluorescent protein were injected stereotactically into the right cerebral hemisphere of nude mice. Moribund mice were killed and autopsied, and the brain was evaluated by fluorescence imaging or histological examination. RESULTS: Intracerebral inoculation of melanoma cells produced multiple lesions involving all regions of the brain, suggesting that the cells were able to migrate over substantial distances within the brain. Multiple modes of transport were identified, and all transport modes were observed in all four melanoma lines. Thus, the melanoma cells were passively transported via the flow of cerebrospinal fluid in the meninges and ventricles, they migrated actively along leptomeningeal and brain parenchymal blood vessels, and they migrated actively along the surfaces separating different brain compartments. CONCLUSION: Migration of melanoma cells after initial arrest, extravasation, and growth at a single location within the brain may contribute significantly to the development of multiple melanoma brain metastases.

  11. Breaking the Blood-Brain Barrier With Mannitol to Aid Stem Cell Therapeutics in the Chronic Stroke Brain.

    Science.gov (United States)

    Tajiri, Naoki; Lee, Jea Young; Acosta, Sandra; Sanberg, Paul R; Borlongan, Cesar V

    2016-01-01

    Blood-brain barrier (BBB) permeabilizers, such as mannitol, can facilitate peripherally delivered stem cells to exert therapeutic benefits on the stroke brain. Although this BBB permeation-aided stem cell therapy has been demonstrated in the acute stage of stroke, such BBB permeation in the chronic stage of the disease remains to be examined. Adult Sprague-Dawley rats initially received sham surgery or experimental stroke via the 1-h middle cerebral artery occlusion (MCAo) model. At 1 month after the MCAo surgery, stroke animals were randomly assigned to receive human umbilical cord stem cells only (2 million viable cells), mannitol only (1.1 mol/L mannitol at 4°C), combined human umbilical cord stem cells (200,000 viable cells) and mannitol (1.1 mol/L mannitol at 4°C), and vehicle (phosphate-buffered saline) only. Stroke animals that received human umbilical cord blood cells alone or combined human umbilical cord stem cells and mannitol exhibited significantly improved motor performance and significantly better brain cell survival in the peri-infarct area compared to stroke animals that received vehicle or mannitol alone, with mannitol treatment reducing the stem cell dose necessary to afford functional outcomes. Enhanced neurogenesis in the subventricular zone accompanied the combined treatment of human umbilical cord stem cells and mannitol. We showed that BBB permeation facilitates the therapeutic effects of a low dose of peripherally transplanted stem cells to effectively cause functional improvement and increase neurogenesis in chronic stroke.

  12. Feasibility and Efficiency of Human Bone Marrow Stromal Cell Culture with Allogeneic Platelet Lysate-Supplementation for Cell Therapy against Stroke

    Directory of Open Access Journals (Sweden)

    Chengbo Tan

    2016-01-01

    Full Text Available Currently, there is increasing interest in human bone marrow stromal cells (hBMSCs as regeneration therapy against cerebral stroke. The aim of the present study was to evaluate the feasibility and validity of hBMSC cultures with allogeneic platelet lysates (PLs. Platelet concentrates (PC were harvested from healthy volunteers and made into single donor-derived PL (sPL. The PL mixtures (mPL were made from three different sPL. Some growth factors and platelet cell surface antigens were detected by enzyme-linked immunosorbent assay (ELISA. The hBMSCs cultured with 10% PL were analyzed for their proliferative potential, surface markers, and karyotypes. The cells were incubated with superparamagnetic iron oxide (SPIO agents and injected into a pig brain. MRI and histological analysis were performed. Consequently, nine lots of sPL and three mPL were prepared. ELISA analysis showed that PL contained adequate growth factors and a particle of platelet surface antigens. Cell proliferation capacity of PLs was equivalent to or higher than that of fetal calf serum (FCS. No contradiction in cell surface markers and no chromosomal aberrations were found. The MRI detected the distribution of SPIO-labeled hBMSCs in the pig brain. In summary, the hBMSCs cultured with allogeneic PL are suitable for cell therapy against stroke.

  13. Possible promotion of neuronal differentiation in fetal rat brain neural progenitor cells after sustained exposure to static magnetism.

    Science.gov (United States)

    Nakamichi, Noritaka; Ishioka, Yukichi; Hirai, Takao; Ozawa, Shusuke; Tachibana, Masaki; Nakamura, Nobuhiro; Takarada, Takeshi; Yoneda, Yukio

    2009-08-15

    We have previously shown significant potentiation of Ca(2+) influx mediated by N-methyl-D-aspartate receptors, along with decreased microtubules-associated protein-2 (MAP2) expression, in hippocampal neurons cultured under static magnetism without cell death. In this study, we investigated the effects of static magnetism on the functionality of neural progenitor cells endowed to proliferate for self-replication and differentiate into neuronal, astroglial, and oligodendroglial lineages. Neural progenitor cells were isolated from embryonic rat neocortex and hippocampus, followed by culture under static magnetism at 100 mT and subsequent determination of the number of cells immunoreactive for a marker protein of particular progeny lineages. Static magnetism not only significantly decreased proliferation of neural progenitor cells without affecting cell viability, but also promoted differentiation into cells immunoreactive for MAP2 with a concomitant decrease in that for an astroglial marker, irrespective of the presence of differentiation inducers. In neural progenitors cultured under static magnetism, a significant increase was seen in mRNA expression of several activator-type proneural genes, such as Mash1, Math1, and Math3, together with decreased mRNA expression of the repressor type Hes5. These results suggest that sustained static magnetism could suppress proliferation for self-renewal and facilitate differentiation into neurons through promoted expression of activator-type proneural genes by progenitor cells in fetal rat brain.

  14. A retinoic acid-enhanced, multicellular human blood-brain barrier model derived from stem cell sources

    Science.gov (United States)

    Lippmann, Ethan S.; Al-Ahmad, Abraham; Azarin, Samira M.; Palecek, Sean P.; Shusta, Eric V.

    2014-02-01

    Blood-brain barrier (BBB) models are often used to investigate BBB function and screen brain-penetrating therapeutics, but it has been difficult to construct a human model that possesses an optimal BBB phenotype and is readily scalable. To address this challenge, we developed a human in vitro BBB model comprising brain microvascular endothelial cells (BMECs), pericytes, astrocytes and neurons derived from renewable cell sources. First, retinoic acid (RA) was used to substantially enhance BBB phenotypes in human pluripotent stem cell (hPSC)-derived BMECs, particularly through adherens junction, tight junction, and multidrug resistance protein regulation. RA-treated hPSC-derived BMECs were subsequently co-cultured with primary human brain pericytes and human astrocytes and neurons derived from human neural progenitor cells (NPCs) to yield a fully human BBB model that possessed significant tightness as measured by transendothelial electrical resistance (~5,000 Ωxcm2). Overall, this scalable human BBB model may enable a wide range of neuroscience studies.

  15. Characterizing parameters of Jatropha curcas cell cultures for microgravity studies

    Science.gov (United States)

    Vendrame, Wagner A.; Pinares, Ania

    2013-06-01

    Jatropha (Jatropha curcas) is a tropical perennial species identified as a potential biofuel crop. The oil is of excellent quality and it has been successfully tested as biodiesel and in jet fuel mixes. However, studies on breeding and genetic improvement of jatropha are limited. Space offers a unique environment for experiments aiming at the assessment of mutations and differential gene expression of crops and in vitro cultures of plants are convenient for studies of genetic variation as affected by microgravity. However, before microgravity studies can be successfully performed, pre-flight experiments are necessary to characterize plant material and validate flight hardware environmental conditions. Such preliminary studies set the ground for subsequent spaceflight experiments. The objectives of this study were to compare the in vitro growth of cultures from three explant sources (cotyledon, leaf, and stem sections) of three jatropha accessions (Brazil, India, and Tanzania) outside and inside the petriGAP, a modified group activation pack (GAP) flight hardware to fit petri dishes. In vitro jatropha cell cultures were established in petri dishes containing a modified MS medium and maintained in a plant growth chamber at 25 ± 2 °C in the dark. Parameters evaluated were surface area of the explant tissue (A), fresh weight (FW), and dry weight (DW) for a period of 12 weeks. Growth was observed for cultures from all accessions at week 12, including subsequent plantlet regeneration. For all accessions differences in A, FW and DW were observed for inside vs. outside the PetriGAPs. Growth parameters were affected by accession (genotype), explant type, and environment. The type of explant influenced the type of cell growth and subsequent plantlet regeneration capacity. However, overall cell growth showed no abnormalities. The present study demonstrated that jatropha in vitro cell cultures are suitable for growth inside PetriGAPs for a period of 12 weeks. The parameters

  16. Uptake and Transport of Superparamagnetic Iron Oxide Nanoparticles through Human Brain Capillary Endothelial Cells

    NARCIS (Netherlands)

    Thomsen, L.B.; Linemann, T.; Pondman, K.M.; Lichota, J.; Kim, K.S.; Pieters, R.J.; Visser, G.M.; Moos, T.

    2013-01-01

    The blood–brain barrier (BBB) formed by brain capillary endothelial cells (BCECs) constitutes a firm physical, chemical, and immunological barrier, making the brain accessible to only a few percent of potential drugs intended for treatment inside the central nervous system. With the purpose of

  17. Aging and senescence of skin cells in culture

    DEFF Research Database (Denmark)

    Rattan, Suresh

    2015-01-01

    Studying age-related changes in the physiology, biochemistry, and molecular biology of isolated skin cell populations in culture has greatly expanded the understanding of the fundamental aspects of skin aging. The three main cell types that have been studied extensively with respect to cellular...... aging in vitro are dermal fibroblasts, epidermal keratinocytes, and melanocytes. Serial subcultivation of normal diploid skin cells can be performed only a limited number of times, and the emerging senescent phenotype can be categorized into structural, physiological, biochemical, and molecular...... phenotypes, which can be used as biomarkers of cellular aging in vitro. The rate and phenotype of aging are different in different cell types. There are both common features and specific features of aging of skin fibroblasts, keratinocytes, melanocytes, and other cell types. A progressive accumulation...

  18. Co-culture with Sertoli cells promotes proliferation and migration of umbilical cord mesenchymal stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Fenxi, E-mail: fxzhang0824@gmail.com [Department of Anatomy, Sanquan College, Xinxiang Medical University, Henan 453003, People' s Republic of China (China); Hong, Yan; Liang, Wenmei [Department of Histology and Embryology, Guiyang Medical University, Guizhou 550004, People' s Republic of China (China); Ren, Tongming [Department of Anatomy, Sanquan College, Xinxiang Medical University, Henan 453003, People' s Republic of China (China); Jing, Suhua [ICU Center, The Third Hospital of Xinxiang Medical University, Henan 453003, People' s Republic of China (China); Lin, Juntang [Stem Cell Center, Xinxiang Medical University, Henan 453003, People' s Republic of China (China)

    2012-10-12

    Highlights: Black-Right-Pointing-Pointer Co-culture of Sertoli cells (SCs) with human umbilical cord mesenchymal stem cells (UCMSCs). Black-Right-Pointing-Pointer Presence of SCs dramatically increased proliferation and migration of UCMSCs. Black-Right-Pointing-Pointer Presence of SCs stimulated expression of Mdm2, Akt, CDC2, Cyclin D, CXCR4, MAPKs. -- Abstract: Human umbilical cord mesenchymal stem cells (hUCMSCs) have been recently used in transplant therapy. The proliferation and migration of MSCs are the determinants of the efficiency of MSC transplant therapy. Sertoli cells are a kind of 'nurse' cells that support the development of sperm cells. Recent studies show that Sertoli cells promote proliferation of endothelial cells and neural stem cells in co-culture. We hypothesized that co-culture of UCMSCs with Sertoli cells may also promote proliferation and migration of UCMSCs. To examine this hypothesis, we isolated UCMSCs from human cords and Sertoli cells from mouse testes, and co-cultured them using a Transwell system. We found that UCMSCs exhibited strong proliferation ability and potential to differentiate to other cell lineages such as osteocytes and adipocytes. The presence of Sertoli cells in co-culture significantly enhanced the proliferation and migration potential of UCMSCs (P < 0.01). Moreover, these phenotypic changes were accompanied with upregulation of multiple genes involved in cell proliferation and migration including phospho-Akt, Mdm2, phospho-CDC2, Cyclin D1, Cyclin D3 as well as CXCR4, phospho-p44 MAPK and phospho-p38 MAPK. These findings indicate that Sertoli cells boost UCMSC proliferation and migration potential.

  19. Co-culture with Sertoli cells promotes proliferation and migration of umbilical cord mesenchymal stem cells

    International Nuclear Information System (INIS)

    Zhang, Fenxi; Hong, Yan; Liang, Wenmei; Ren, Tongming; Jing, Suhua; Lin, Juntang

    2012-01-01

    Highlights: ► Co-culture of Sertoli cells (SCs) with human umbilical cord mesenchymal stem cells (UCMSCs). ► Presence of SCs dramatically increased proliferation and migration of UCMSCs. ► Presence of SCs stimulated expression of Mdm2, Akt, CDC2, Cyclin D, CXCR4, MAPKs. -- Abstract: Human umbilical cord mesenchymal stem cells (hUCMSCs) have been recently used in transplant therapy. The proliferation and migration of MSCs are the determinants of the efficiency of MSC transplant therapy. Sertoli cells are a kind of “nurse” cells that support the development of sperm cells. Recent studies show that Sertoli cells promote proliferation of endothelial cells and neural stem cells in co-culture. We hypothesized that co-culture of UCMSCs with Sertoli cells may also promote proliferation and migration of UCMSCs. To examine this hypothesis, we isolated UCMSCs from human cords and Sertoli cells from mouse testes, and co-cultured them using a Transwell system. We found that UCMSCs exhibited strong proliferation ability and potential to differentiate to other cell lineages such as osteocytes and adipocytes. The presence of Sertoli cells in co-culture significantly enhanced the proliferation and migration potential of UCMSCs (P < 0.01). Moreover, these phenotypic changes were accompanied with upregulation of multiple genes involved in cell proliferation and migration including phospho-Akt, Mdm2, phospho-CDC2, Cyclin D1, Cyclin D3 as well as CXCR4, phospho-p44 MAPK and phospho-p38 MAPK. These findings indicate that Sertoli cells boost UCMSC proliferation and migration potential.

  20. Endothelial cells stimulate growth of normal and cancerous breast epithelial cells in 3D culture

    Directory of Open Access Journals (Sweden)

    Magnusson Magnus K

    2010-07-01

    Full Text Available Abstract Background Epithelial-stromal interaction provides regulatory signals that maintain correct histoarchitecture and homeostasis in the normal breast and facilitates tumor progression in breast cancer. However, research on the regulatory role of the endothelial component in the normal and malignant breast gland has largely been neglected. The aim of the study was to investigate the effects of endothelial cells on growth and differentiation of human breast epithelial cells in a three-dimensional (3D co-culture assay. Methods Breast luminal and myoepithelial cells and endothelial cells were isolated from reduction mammoplasties. Primary cells and established normal and malignant breast cell lines were embedded in reconstituted basement membrane in direct co-culture with endothelial cells and by separation of Transwell filters. Morphogenic and phenotypic profiles of co-cultures was evaluated by phase contrast microscopy, immunostaining and confocal microscopy. Results In co-culture, endothelial cells stimulate proliferation of both luminal- and myoepithelial cells. Furthermore, endothelial cells induce a subpopulation of luminal epithelial cells to form large acini/ducts with a large and clear lumen. Endothelial cells also stimulate growth and cloning efficiency of normal and malignant breast epithelial cell lines. Transwell and gradient co-culture studies show that endothelial derived effects are mediated - at least partially - by soluble factors. Conclusion Breast endothelial cells - beside their role in transporting nutrients and oxygen to tissues - are vital component of the epithelial microenvironment in the breast and provide proliferative signals to the normal and malignant breast epithelium. These growth promoting effects of endothelial cells should be taken into consideration in breast cancer biology.

  1. Effect of low dose laser on the chorioallantoic culture of retinal pigment cells

    International Nuclear Information System (INIS)

    Yew, D.T.; Lam, S.T.L.; Chan, Y.W.

    1982-01-01

    Low dose laser effects were analysed in chorioallantoic cultures of retinal pigment cells. Decrease in cell sizes and increase in number of mitosis were observed in the experimental cultures. On the other hand, pyknosis did not change significantly following irradiation. Most cells in the control and experimental cultures formed groups. However, 2 types of detached cells were evident. The percentage of detached cells was higher in the experimental culture. (Auth.)

  2. Differential transgene expression in brain cells in vivo and in vitro from AAV-2 vectors with small transcriptional control units

    International Nuclear Information System (INIS)

    Kuegler, S.; Lingor, P.; Schoell, U.; Zolotukhin, S.; Baehr, M.

    2003-01-01

    Adeno-associated- (AAV) based vectors are promising tools for gene therapy applications in several organs, including the brain, but are limited by their small genome size. Two short promoters, the human synapsin 1 gene promoter (hSYN) and the murine cytomegalovirus immediate early promoter (mCMV), were evaluated in bicistronic AAV-2 vectors for their expression profiles in cultured primary brain cells and in the rat brain. Whereas transgene expression from the hSYN promoter was exclusively neuronal, the murine CMV promoter targeted expression mainly to astrocytes in vitro and showed weak transgene expression in vivo in retinal and cortical neurons, but strong expression in thalamic neurons. We propose that neuron specific transgene expression in combination with enhanced transgene capacity will further substantially improve AAV based vector technology

  3. Scale-up of cell culture bioreactors using biomechatronic design.

    Science.gov (United States)

    Mandenius, Carl-Fredrik; Björkman, Mats

    2012-08-01

    Scale-up of cell culture bioreactors is a challenging engineering work that requires wide competence in cell biology, mechanical engineering and bioprocess design. In this article, a new approach for cell culture bioreactor scale-up is suggested that is based on biomechatronic design methodology. The approach differs from traditional biochemical engineering methodology by applying a sequential design procedure where the needs of the users and alternative design solutions are systematically analysed. The procedure is based on the biological and technical functions of the scaled-up bioreactor that are derived in functional maps, concept generation charts and scoring and interaction matrices. Basic reactor engineering properties, such as mass and heat transfer and kinetics are integrated in the procedure. The methodology results in the generation of alternative design solutions that are thoroughly ranked with help of the user needs. Examples from monoclonal antibodies and recombinant protein production illuminate the steps of the procedure. The methodology provides engineering teams with additional tools that can significantly facilitate the design of new production methods for cell culture processes. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. 3D Cell Culture Imaging with Digital Holographic Microscopy

    Science.gov (United States)

    Dimiduk, Thomas; Nyberg, Kendra; Almeda, Dariela; Koshelva, Ekaterina; McGorty, Ryan; Kaz, David; Gardel, Emily; Auguste, Debra; Manoharan, Vinothan

    2011-03-01

    Cells in higher organisms naturally exist in a three dimensional (3D) structure, a fact sometimes ignored by in vitro biological research. Confinement to a two dimensional culture imposes significant deviations from the native 3D state. One of the biggest obstacles to wider use of 3D cultures is the difficulty of 3D imaging. The confocal microscope, the dominant 3D imaging instrument, is expensive, bulky, and light-intensive; live cells can be observed for only a short time before they suffer photodamage. We present an alternative 3D imaging techinque, digital holographic microscopy, which can capture 3D information with axial resolution better than 2 μm in a 100 μm deep volume. Capturing a 3D image requires only a single camera exposure with a sub-millisecond laser pulse, allowing us to image cell cultures using five orders of magnitude less light energy than with confocal. This can be done with hardware costing ~ 1000. We use the instrument to image growth of MCF7 breast cancer cells and p. pastoras yeast. We acknowledge support from NSF GRFP.

  5. Individualized medicine for renal cell carcinoma: establishment of primary cell line culture from surgical specimens.

    Science.gov (United States)

    Kim, Fernando J; Campagna, Adriano; Khandrika, Lakshmipathi; Koul, Sweaty; Byun, Seok-Soo; vanBokhoven, Adrie; Moore, Ernest E; Koul, Hari

    2008-10-01

    The lack of effective "in vivo" and "in vitro" models to predict success of pharmacological therapy for patients with renal cell carcinoma, as well as, the variety of cancer cell types demands the development of better experimental models to understand the pathophysiology of the disease and evaluate drug sensitivity in vitro. To develop primary renal cancer cell culture irrespective of tumor grade and tumor type, harvested from the patient's pathological specimen immediately after the laparoscopic radical nephrectomy to study potential "in vivo" pharmacological sensitivity. A total of 24 patients (17 males and 7 females). Mean age of 63.1+/-3.1 y.o. The mean size of the renal masses was 7.56+/-3.1 cm. Normal and pathological renal tissue was collected immediately after the specimen was extracted and submitted to enzymatic digestion for 16-24 hours. Clear cell carcinoma cells were selected through multiple passages in DMEM medium supplemented with glucose and antibiotics. Establishment of cell line culture from all the patients' specimens irrespective of tumor grade and tumor type was achieved successfully. In addition to the tumor cell line culture, normal parenchyma tissue yielded primary cell lines to allow testing the response of tumor types to various pharmacological therapeutic agents and toxicity of such treatments to healthy tissue. From the initial collection of the specimens obtained after the removal of the kidney to the development of cell lines took occurred in average 32+6 hrs. The cells in culture showed characteristics of epithelial cells; like expression on cytokeratin and were maintained in culture for more than 20 passages. The development of renal cancer cell cultures in vitro is labor intense but may yield a more realistic model to tailor pharmacological therapies and predict therapeutic success prior to "in vivo" application-a step in the direction of individualized medicine for RCC.

  6. Cell division in Escherichia coli cultures monitored at single cell resolution

    Directory of Open Access Journals (Sweden)

    Luidalepp Hannes

    2008-04-01

    Full Text Available Abstract Background A fundamental characteristic of cells is the ability to divide. To date, most parameters of bacterial cultures, including cell division, have been measured as cell population averages, assuming that all bacteria divide at a uniform rate. Results We monitored the division of individual cells in Escherichia coli cultures during different growth phases. Our experiments are based on the dilution of green fluorescent protein (GFP upon cell division, monitored by flow cytometry. The results show that the vast majority of E. coli cells in exponentially growing cultures divided uniformly. In cultures that had been in stationary phase up to four days, no cell division was observed. However, upon dilution of stationary phase culture into fresh medium, two subpopulations of cells emerged: one that started dividing and another that did not. These populations were detectable by GFP dilution and displayed different side scatter parameters in flow cytometry. Further analysis showed that bacteria in the non-growing subpopulation were not dead, neither was the difference in growth capacity reducible to differences in stationary phase-specific gene expression since we observed uniform expression of several stress-related promoters. The presence of non-growing persisters, temporarily dormant bacteria that are tolerant to antibiotics, has previously been described within growing bacterial populations. Using the GFP dilution method combined with cell sorting, we showed that ampicillin lyses growing bacteria while non-growing bacteria retain viability and that some of them restart growth after the ampicillin is removed. Thus, our method enables persisters to be monitored even in liquid cultures of wild type strains in which persister formation has low frequency. Conclusion In principle, the approaches developed here could be used to detect differences in cell division in response to different environmental conditions and in cultures of unicellular

  7. Cell proliferation and apoptosis in optic nerve and brain integration centers of adult trout Oncorhynchus mykiss after optic nerve injury

    Directory of Open Access Journals (Sweden)

    Evgeniya V Pushchina

    2016-01-01

    Full Text Available Fishes have remarkable ability to effectively rebuild the structure of nerve cells and nerve fibers after central nervous system injury. However, the underlying mechanism is poorly understood. In order to address this issue, we investigated the proliferation and apoptosis of cells in contralateral and ipsilateral optic nerves, after stab wound injury to the eye of an adult trout Oncorhynchus mykiss. Heterogenous population of proliferating cells was investigated at 1 week after injury. TUNEL labeling gave a qualitative and quantitative assessment of apoptosis in the cells of optic nerve of trout 2 days after injury. After optic nerve injury, apoptotic response was investigated, and mass patterns of cell migration were found. The maximal concentration of apoptotic bodies was detected in the areas of mass clumps of cells. It is probably indicative of massive cell death in the area of high phagocytic activity of macrophages/microglia. At 1 week after optic nerve injury, we observed nerve cell proliferation in the trout brain integration centers: the cerebellum and the optic tectum. In the optic tectum, proliferating cell nuclear antigen (PCNA-immunopositive radial glia-like cells were identified. Proliferative activity of nerve cells was detected in the dorsal proliferative (matrix area of the cerebellum and in parenchymal cells of the molecular and granular layers whereas local clusters of undifferentiated cells which formed neurogenic niches were observed in both the optic tectum and cerebellum after optic nerve injury. In vitro analysis of brain cells of trout showed that suspension cells compared with monolayer cells retain higher proliferative activity, as evidenced by PCNA immunolabeling. Phase contrast observation showed mitosis in individual cells and the formation of neurospheres which gradually increased during 1-4 days of culture. The present findings suggest that trout can be used as a novel model for studying neuronal regeneration.

  8. Cell proliferation and apoptosis in optic nerve and brain integration centers of adult trout Oncorhynchus mykiss after optic nerve injury

    Science.gov (United States)

    Pushchina, Evgeniya V.; Shukla, Sachin; Varaksin, Anatoly A.; Obukhov, Dmitry K.

    2016-01-01

    Fishes have remarkable ability to effectively rebuild the structure of nerve cells and nerve fibers after central nervous system injury. However, the underlying mechanism is poorly understood. In order to address this issue, we investigated the proliferation and apoptosis of cells in contralateral and ipsilateral optic nerves, after stab wound injury to the eye of an adult trout Oncorhynchus mykiss. Heterogenous population of proliferating cells was investigated at 1 week after injury. TUNEL labeling gave a qualitative and quantitative assessment of apoptosis in the cells of optic nerve of trout 2 days after injury. After optic nerve injury, apoptotic response was investigated, and mass patterns of cell migration were found. The maximal concentration of apoptotic bodies was detected in the areas of mass clumps of cells. It is probably indicative of massive cell death in the area of high phagocytic activity of macrophages/microglia. At 1 week after optic nerve injury, we observed nerve cell proliferation in the trout brain integration centers: the cerebellum and the optic tectum. In the optic tectum, proliferating cell nuclear antigen (PCNA)-immunopositive radial glia-like cells were identified. Proliferative activity of nerve cells was detected in the dorsal proliferative (matrix) area of the cerebellum and in parenchymal cells of the molecular and granular layers whereas local clusters of undifferentiated cells which formed neurogenic niches were observed in both the optic tectum and cerebellum after optic nerve injury. In vitro analysis of brain cells of trout showed that suspension cells compared with monolayer cells retain higher proliferative activity, as evidenced by PCNA immunolabeling. Phase contrast observation showed mitosis in individual cells and the formation of neurospheres which gradually increased during 1–4 days of culture. The present findings suggest that trout can be used as a novel model for studying neuronal regeneration. PMID:27212918

  9. Calcium exchange, structure, and function in cultured adult myocardial cells

    International Nuclear Information System (INIS)

    Langer, G.A.; Frank, J.S.; Rich, T.L.; Orner, F.B.

    1987-01-01

    Cells digested from adult rat heart and cultured for 14 days demonstrate all the structural elements, in mature form, associated with the process of excitation-contraction (EC) coupling. The transverse tubular (TT) system is well developed with an extensive junctional sarcoplasmic reticulum (JSR). In nonphosphate-containing buffer contraction of the cells is lost as rapidly as zero extracellular Ca concentration ([Ca] 0 ) solution is applied and a negative contraction staircase is produced on increase of stimulation frequency. Structurally and functionally the cells have the characteristics of adult cells in situ. 45 Ca exchange and total 45 Ca measurement in N-2-hydroxyethylpiperazine N'-2-ethanesulfonic acid (HEPES)-buffered perfusate define three components of cellular Ca: 1) a rapidly exchangeable component accounting for 36% of total Ca, 2) a slowly exchangeable component (t/sub 1/2/ 53 min) accounting for 7% total Ca, and 3) the remaining 57% cellular Ca is inexchangeable (demonstrates no significant exchange within 60 min). The slowly exchangeable component can be increased 10-fold within 60 min by addition of phosphate to the perfusate. The Ca distribution and exchange characteristics are little different from those of 3-day cultures of neonatal rat heart previously studied. The results suggest that the cells are representative of adult cells in situ and that both sarcolemmal-bound and sarcoplasmic reticular Ca contribute to the component of Ca that is rapidly exchangeable

  10. Biofunctionalized Plants as Diverse Biomaterials for Human Cell Culture.

    Science.gov (United States)

    Fontana, Gianluca; Gershlak, Joshua; Adamski, Michal; Lee, Jae-Sung; Matsumoto, Shion; Le, Hau D; Binder, Bernard; Wirth, John; Gaudette, Glenn; Murphy, William L

    2017-04-01

    The commercial success of tissue engineering products requires efficacy, cost effectiveness, and the possibility of scaleup. Advances in tissue engineering require increased sophistication in the design of biomaterials, often challenging the current manufacturing techniques. Interestingly, several of the properties that are desirable for biomaterial design are embodied in the structure and function of plants. This study demonstrates that decellularized plant tissues can be used as adaptable scaffolds for culture of human cells. With simple biofunctionalization technique, it is possible to enable adhesion of human cells on a diverse set of plant tissues. The elevated hydrophilicity and excellent water transport abilities of plant tissues allow cell expansion over prolonged periods of culture. Moreover, cells are able to conform to the microstructure of the plant frameworks, resulting in cell alignment and pattern registration. In conclusion, the current study shows that it is feasible to use plant tissues as an alternative feedstock of scaffolds for mammalian cells. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Single molecule microscopy in 3D cell cultures and tissues.

    Science.gov (United States)

    Lauer, Florian M; Kaemmerer, Elke; Meckel, Tobias

    2014-12-15

    From the onset of the first microscopic visualization of single fluorescent molecules in living cells at the beginning of this century, to the present, almost routine application of single molecule microscopy, the method has well-proven its ability to contribute unmatched detailed insight into the heterogeneous and dynamic molecular world life is composed of. Except for investigations on bacteria and yeast, almost the entire story of success is based on studies on adherent mammalian 2D cell cultures. However, despite this continuous progress, the technique was not able to keep pace with the move of the cell biology community to adapt 3D cell culture